Mammalian cardiomyocytes actively proliferate during embryonic stages, following which they exit their cell cycle after birth, and the exit is maintained. Previously, we showed that two inhibitory systems (the G1-phase inhibitory system: repression of cyclin D1 expression; the M-phase inhibitory system: inhibition of CDK1 activation) maintain the cell cycle exit of mouse adult cardiomyocytes. We also showed that two CDK inhibitors (CKIs), p21(Cip1) and p27(Kip1), regulate the cell cycle exit in a portion of postnatal cardiomyocytes. It remains unknown whether the two inhibitory systems are involved in the cell cycle exit of postnatal cardiomyocytes and whether p21(Cip1) and p27(Kip1) also inhibit entry to M-phase. Here, we showed that more than 40% of cardiomyocytes entered an additional cell cycle by induction of cyclin D1 expression at postnatal stages, but M-phase entry was inhibited in the majority of cardiomyocytes. Marked cell cycle progression and endoreplication were observed in cardiomyocytes of p21(Cip1) knockout mice at 4 weeks of age. In addition, tri- and tetranucleated cardiomyocytes increased significantly in p21(Cip1) knockout mice. These data showed that the G1-phase inhibitory system and two CKIs (p21(Cip1) and p27(Kip1)) inhibit entry to an additional cell cycle in postnatal cardiomyocytes, and that the M-phase inhibitory system and p21(Cip1) inhibit M-phase entry of cardiomyocytes which have entered the additional cell cycle. (C) 2015 Elsevier Inc. All rights reserved.