The signals that regulate cardiac myocyte maturation in the neonatal heart are not completely understood. In our study we examined the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on primary cultures of ventricular myocytes isolated from neonatal rat hearts. Our data show that 1,25-(OH)2D3 inhibited an increase in both the muscle-specific form of creatine kinase and V1 myosin isoenzyme levels in myocytes induced to mature by serum withdrawal. Thus, in contrast to other cell types studied to date, in the heart, 1,25-(OH)2D3 blocks cell maturation. Treating cultures with phorbol 12-myristate 13-acetate induced a decrease in the muscle-specific isoenzyme of creatine kinase similar to the effects of 1,25-(OH)2D3. Interestingly, we found that staurosporine, a protein kinase-C inhibitor, blunts the effects of 1,25-(OH)2D3 and has the opposite effect of phorbol 12-myristate 13-acetate on cultured myocytes induced to mature by serum withdrawal. Thus, our data identify a novel role for 1,25-(OH)2D3 in the regulation of myocardial development and suggest that 1,25-(OH)2D3 may be acting through a protein kinase-dependent mechanism to maintain cardiac myocytes in an immature state.