Myocytes from rabbit ventricle were enzymatically dissociated and the effects of protein kinase C (PKC) on the properties of single ATP-sensitive (KATP) channels were studied using excised inside-out membrane patches. Application of a purified, constitutively active form of PKC (20 nM) to the intracellular surface of inside-out patches caused a 48% +/- 4% (n = 18) reduction in the open probability of single KATP channels. In the presence of the PKC inhibitors peptide PKC(19-31) or chelerythrine chloride, PKC had no effect on KATP channel properties. Heat-inactivated PKC had no effect on channel properties. KATP channel activity returned spontaneously after removal of PKC. However, application of okadaic acid, at a concentration (5 nM) appropriate for specific inhibition of type 2A protein phosphatase (PP-2A), after removal of PKC, prevented spontaneous recovery of channel activity. Treatment with purified PP-2A during the PKC-mediated inhibition of KATP channel activity caused a partial or full restoration of activity. The Hill coefficient for ATP binding was reduced from 2.2 (control) to 1.2 in the presence of PKC. The apparent inhibition constant (Ki) for ATP was unaffected by PKC [Ki(control) = 21 microM; Ki(PKC) = 20 microM]. PKC is, therefore, capable of inhibiting cardiac KATP channel activity, and the extent to which the channels remain phosphorylated appears to be dependent on membrane-associated PP-2A activity. These enzymes may, therefore, be involved in signal transduction mechanisms which serve to regulate the activity of cardiac KATP channels.