Protein kinase C (PKC) enzymes regulate numerous cardiac functions. In the present study, we determined the effects of the PKC-activating drug 4-beta phorbol 12-myristate 13-acetate (4-beta PMA) on the rate of contraction and correlated these changes with the distribution and levels of alpha-, beta-, delta-, epsilon-, and zeta-PKC isozymes by using neonatal rat cardiac myocytes in culture. Treatment with 0.3 to 100 nmol/L 4-beta PMA caused negative chronotropic effects on contraction. This effect was maximal at a concentration of 3 nmol/L 4-beta PMA and correlated with redistribution of the alpha- and epsilon-PKC isozymes from the cytosolic to the particulate cell fraction. After a 1-hour treatment with 100 nmol/L PMA, the alpha- and beta-PKC isozymes and an 80-kD zeta-like PKC isozyme were greatly diminished (downregulated), yet the negative chronotropic effect was sustained. Therefore, our results are most consistent with a role for the epsilon-PKC isozyme in suppressing the contraction rate of neonatal rat cardiac myocytes. Understanding the role(s) of individual PKC isozymes in the modulation of cardiac functions may ultimately yield more selective targets for therapies of cardiac disorders.