A card is said to be in play if it is in the play area—the location cards are put when you play them. Under ordinary circumstances, a card enters the play area when you play it, and leaves play when discarded during the Clean-up phase of that turn. Reserve cards also enter the play area when you call them.
There are a handful of cards, the Command variants, which instruct you to play a card without putting it in play. Some Throne Room variants, which cause you to play the same card multiple times, can replay a card even if the first play of the same card caused it to be removed from the play area. In other cases, if a card cannot be moved into the play area (usually as a result of the stop-moving rule), it cannot be played.
Ordinarily cards leave the play area by being discarded during the Clean-up phase of the turn on which they enter play, though several exceptions exist:
- Duration cards, and Command variants playing Duration cards, remain in play until the Clean-up phase of the last turn on which they are expected to have an effect; Throne Room variants playing Duration cards remain in play until the Clean-up phase of the turn when the Duration leaves play.
- One-shots, Reserve cards, and certain other cards have instructions to remove themselves from play as a result of playing them.
- Some cards and card-shaped things have abilities that cause you to remove other cards from play before the Clean-up phase, or do something else with them in the Clean-up phase instead of discarding them: for example, Crypt sets aside Treasure cards from play when you play it, and Improve trashes a card from play during Clean-up instead of discarding it.
Some cards and card-shaped things have abilities that depend on what is in play at a given time. For example, Peddler changes its own cost depending on how many Action cards are in play during the Buy phase, and Changeling can only gain copies of cards that are in play.
 While-in-play abilities
Between Seaside in 2009 and Nocturne in 2017, a number of Dominion cards were released with abilities that are activated while the card is in play, listed below a horizontal dividing line in the card text; these may be contrasted with "on-play" (or "above-the-line") abilities, which are triggered by the card being played, rather than by it being located in the play area. Cards with while-in-play abilities were typically those with persistent effects on the game state that lasted for a period of time; Donald X. Vaccarino's rationale in introducing the while-in-play mechanic was to facilitate tracking of these abilities, so players could simply inspect the cards that were visible in the play area to determine which abilities were in effect.
However, players often found the rules for while-in-play abilities, and the differences between them and on-play abilities, confusing and counterintuitive—especially in cases where the same ability (such as cost reduction) was on-play for some cards (e.g., Bridge) and while-in-play for others (e.g., Highway). As a result of this confusion, Donald X. eventually stopped introducing while-in-play abilities on new cards; and in the 2022 updates and revisions, all remaining cards with while-in-play abilities were either removed to be replaced with new cards in the second edition of Prosperity, or were revised to move the abilities above the line.
 Differences between while-in-play and on-play
Since while-in-play abilities begin to take effect when you play the card, it is easy to mistake them for abilities that are triggered by playing the card. However, while-in-play effects differ from on-play effects in several ways:
- If you play the same card multiple times on the same turn via a Throne Room variant, its "on-play" abilities happen each time you play it; but there is only one copy of the card in the play area, so its while-in-play abilities only happen once.
- If a card is played by a Command variant, it never enters play, so its while-in-play abilities never activate but its on-play effects do. Similarly, if a card is removed from play prematurely, its while-in-play abilities cease to operate, but any persistent on-play abilities remain in effect.
- Enchantress and Ways overrule the on-play abilities of an Action card, but leave the while-in-play abilities unchanged.
 List of cards with while-in-play abilities
The following were revised in 2022 to turn the while-in-play abilities into on-play abilities:
- Seaside: Lighthouse
- Prosperity: Quarry, Hoard
- Cornucopia: Princess
- Hinterlands: Haggler, Highway
- Guilds: Merchant Guild
- Adventures: Bridge Troll
- Empires: Groundskeeper
- Nocturne: Tracker
- Promo: Sauna
 Donald X.'s evolving thoughts on while-in-play
Donald X. originally introduced while-in-play abilities for the sake of tracking persistent effects:
At one point he contemplated revising earlier cards such as Bridge to use while-in-play wording, though he did not end up making this change.
Later, he discovered that players find while-in-play abilities more confusing than on-play abilities, and stopped using while-in-play abilities on new cards:
Finally, in 2022, all cards with "while this is in play" received errata to "this turn."