Mammalian cardiomyocytes actively proliferate during embryonic stages, following which cardiomyocytes exit their cell cycle after birth. The irreversible cell cycle exit inhibits cardiac regeneration by the proliferation of pre-existing cardiomyocytes. Exactly how the cell cycle exit occurs remains largely unknown. Previously, we showed that cyclin E- and cyclin A-CDK activities are inhibited before the CDKs levels decrease in postnatal stages. This result suggests that factors such as CDK inhibitors (CKIs) inhibit CDK activities, and contribute to the cell cycle exit. In the present study, we focused on a Cip/Kip family, which can inhibit cyclin E- and cyclin A-CDK activities. Expression of p21(Cip1) and p27(Kip1) but not p57(Kip2) showed a peak around postnatal day 5, when cyclin E- and cyclin A-CDK activities start to decrease. p21(Cip1) and p27(Kip1) bound to cyclin E, cyclin A and CDK2 at postnatal stages. Cell cycle distribution patterns of postnatal cardiomyocytes in p21(Cip1) and p27(Kip1) knockout mice showed failure in the cell cycle exit at G1-phase, and endoreplication. These results indicate that p21(Cip1) and p27(Kip) play important roles in the cell cycle exit of postnatal cardiomyocytes. (C) 2013 Elsevier Inc. All rights reserved.