Both protein kinase C (PKC) and adenosine receptor activation have been shown to enhance ATP-sensitive K+ (KATP) channels. The present studies were designed to determine whether PKC mediates adenosine effects on the KATP channel. The dependence of KATP channel activity (nPo) on intracellular ATP concentration ([ATP]i) was determined in excised rabbit ventricular membrane patches. External adenosine (100 microM in the pipette solution) significantly increased KATP nPo at all [ATP]i between 5 and 50 microM by decreasing channel sensitivity to [ATP]i (dissociation constant increased from 7.4 +/- 0.8 to 22.2 +/- 3.1 microM, P < 0.001), an effect blocked by the adenosine receptor antagonist 8-phenyltheophylline (10 microM). When the highly selective PKC blocker bisindolylmaleimide (BIM) was included in the internal (bath) solution, the KATP-stimulating action of adenosine was prevented. The addition of BIM to the superfusate rapidly inhibited KATP channels activated by adenosine. Endogenous PKC activation by phorbol 12,13-didecanoate (PDD), but not administration of the inactive congener 4alpha-PDD, enhanced KATP activity. Internal guanosine 5'-O-(2-thiodiphosphate) prevented KATP activation by adenosine, an effect which could be overridden by exposure to PDD. We conclude that PKC mediates adenosine activation of KATP channels in excised membrane patches in a membrane-delimited fashion.