—The precise role of cell cycle–dependent molecules in controlling the switch from cardiac myocyte hyperplasia to hypertrophy remains to be determined. We report that loss of p27^KIP1 in the mouse results in a significant increase in heart size and in the total number of cardiac myocytes. In comparison to p27^KIP1+/+ myocytes, the percentage of neonatal p27^KIP1−/− myocytes in S phase was increased significantly, concomitant with a significant decrease in the percentage of G₀/G₁ cells. The expressions of proliferating cell nuclear antigen, G₁/S and G₂/M phase–acting cyclins, and cyclin-dependent kinases (CDKs) were upregulated significantly in ventricular tissue obtained from early neonatal p27^KIP1−/− mice, concomitant with a substantial decrease in the expressions of G₁ phase–acting cyclins and CDKs. Furthermore, mRNA expressions of the embryonic genes atrial natriuretic factor and α-skeletal actin were detectable at significant levels in neonatal and adult p27^KIP1−/− mouse hearts but were undetectable in p27^KIP1+/+ hearts. In addition, loss of p27^KIP1 was not compensated for by the upregulation of other CDK inhibitors. Thus, the loss of p27^KIP1 results in prolonged proliferation of the mouse cardiac myocyte and perturbation of myocyte hypertrophy.