Mediators involved in ischemia preconditioning such as adenosine and norepinephrine, can activate protein kinase C (PKC), and a variety of observations suggest that both PKC and ATP-sensitive K+ current (I (KATP) play essential roles in ischemic preconditioning. PKC is therefore a candidate to link receptor binding to I(KATP) activation, but it has not been shown whether and how PKC can activate I(KATP) in the heart. The present study was designed to determine whether PKC can activate I(KATP) in rabbit and human ventricular myocytes. Under conditions designed to minimize Na+ and Ca2+ currents, dialysis of rabbit ventricular myocytes with pipette solutions containing reduced [ATP] elicited I(KATP)++, with a 50% effective concentration (EC50)of 260 micromol/L. In cells that failed to show I (KATP) under control conditions, superfusion with 1 micromol/L phorbol 12,13-didecanoate (PDD) elicited I(KATP) in a fashion that depended on pipette [ATP], with an [ATP] EC 50 of 601 micromol/L. PDD-induced I(KATP) activation was concentration dependent, with an EC 50 of 7.1 nmol/L. The highly selective PKC inhibitor bisindolylmaleimide totally prevented I(KATP) activation by PDD, and in blinded experiments, 1 micromol/L PDD elicited I(KATP) in eight of nine cells, whereas its non-PKC-stimulating analogue 4 alpha-PDD failed to elicit I(KATP) in any of the five cells tested (P = .003). Similar experiments were conducted in human ventricular myocytes and showed that 0.1 micromol/L PDD elicited I( KATP) at pipette [ATP] of 100 and 400 micromol/L (five of five cells at each concentration) but not at 1 mmol/L [ATP] (none of five cells). We conclude that PKC activates I(KATP) in rabbit and human ventricular myocytes by reducing channel sensitivity to intracellular ATP. This finding has potentially important implications for understanding the mechanisms of ischemic preconditioning.