Cyclin-dependent kinase 2 (cdk2) plays a critical role in the G1- to S-phase checkpoint of the cell cycle. Adult cardiomyocytes are believed to withdraw from the cell cycle. To determine whether forced overexpression of cdk2 results in altered cell-cycle regulation in the adult heart, we generated transgenic mice specifically overexpressing cdk2 in hearts. Transgenic hearts expressed high levels of both cdk2 mRNA and catalytically active cdk2 proteins. Cdk2 overexpression significantly increased the levels of cdk4 and cyclins A, D3, and E. There was an increase in both DNA synthesis and proliferating cell nuclear antigen levels in the adult transgenic hearts. The ratio of heart weight to body weight in cdk2 transgenic mice was significantly increased in neonatal day 2 but not in adults compared with that of wild-type mice. Analysis of dispersed individual adult cardiomyocytes showed a 5.6-fold increase in the proportion of smaller mononuclear cardiomyocytes in the transgenic mice. Echocardiography revealed that transgenic heart was functionally normal. However, adult transgenic ventricles expressed beta-myosin heavy chain and atrial natriuretic factor. Surgically induced pressure overload caused an exaggerated maladaptive hypertrophic response in transgenic mice but did not change the proportion of mononuclear cardiomyocytes. The data suggest that overexpression of cdk2 promotes smaller, less-differentiated mononuclear cardiomyocytes in adult hearts that respond in an exaggerated manner to pressure overload.