Ischemic preconditioning has been shown to involve the activation of adenosine receptors, protein kinase C (PKC), and ATP-sensitive K⁺ (K_ATP) channels. We investigated the effects of PKC activation and adenosine on K_ATP current (I_K,ATP) and action potentials in isolated rabbit ventricular myocytes. Responses to pinacidil (100 to 400 μmol/L), an opener of K_ATP channels, were markedly increased by preexposure to the PKC activator phorbol 12-myristate 13-acetate (PMA, 100 nmol/L). I_K,ATP measured at 0 mV was increased by PMA pretreatment from 0.55±0.32 to 3.25±0.47 nA (n=6, P<.01). We next determined whether PKC activation abbreviates the time required to turn on I_K,ATP during metabolic inhibition (MI). In control cells in which MI was induced by 2 mmol/L cyanide and 0 glucose, I_K,ATP developed after an average of 15.1±2.4 minutes (n=8). Ten-minute pretreatment with PMA alone (PMA+MI) did not significantly alter this latency (11.9±2.0 minutes, n=8). Since adenosine receptor activation has been shown to play an important role in the preconditioning response, two groups of myocytes were studied with adenosine (10 μmol/L) included during MI. Without PMA, adenosine alone (MI+Ado) did not affect the latency to develop I_K,ATP (12.3±1.5 minutes, n=8). However, if cells were pretreated with PMA and then subjected to MI in the presence of adenosine (PMA+MI+Ado), the latency was greatly shortened to 5.5±1.6 minutes (n=8; P<.02 versus MI, PMA+MI, and MI+Ado groups). This effect could not be reproduced by an inactive phorbol but was completely abolished by the adenosine receptor antagonist 8-(p-sulfophenyl)-theophylline. The opening of K_ATP channels may be cardioprotective because of the abbreviation of action potential duration (APD) during ischemia. Therefore, we tested whether PKC activation could modify the time course of APD shortening during MI. Consistent with the ionic current measurements, PMA pretreatment significantly accelerated APD shortening, but only when adenosine (10 μmol/L) was included during MI. The effects were not attributable to accelerated ATP consumption: PMA pretreatment did not alter the time required to induce rigor during MI, whether or not adenosine was included. Our results indicate that PKC activation increases the I_K,ATP induced by pinacidil or by MI. The latter effect requires concomitant adenosine receptor activation. The synergistic modulation of I_K,ATP by PKC and adenosine provides an explicit basis for current paradigms of ischemic preconditioning.