It has been suggested that preconditioning (PC) against infarction in the rat heart is mediated by opening of ATP-sensitive potassium (Katp) channels, which may be induced by receptor-triggered activation of protein kinase C (PKC). However, the mechanism of suppression of reperfusion arrhythmias by PC remains unclear. This study first examined whether suppression of reperfusion arrhythmias by PC requires the activation of Katp channels, PKC, and opioid receptors. In anesthetized rats, reperfusion arrhythmias were induced by occluding the left main coronary artery for 5 min and subsequent reperfusion. In untreated control rats, the incidence of reperfusion ventricular tachycardia (VT) was 100%, and 80% of the VT deteriorated to ventricular fibrillation (VF). PC with 2-min ischemia/5-min reperfusion reduced the incidences of VT and VF to 30% and 0, respectively. Although a Katp channel blocker, 5-hydroxydecanoate (5-HD), alone caused no significant effect on the incidence of reperfusion VT, this agent blocked the suppression of reperfusion VT by PC (VT incidence, 91%). The incidence of reperfusion VF in the 5-HD-treated rats tended to be lower than that in the untreated controls and was not different between preconditioned and nonpreconditioned groups (30 vs. 27%). PKC inhibitors staurosporine and calphostin C modified neither reperfusion arrhythmias nor the antiarrhythmic effect of PC on reperfusion VT and VF. Naloxone (6 mg/kg, i.v.) did not alter the incidence or duration of VT in the nonpreconditioned heart. However, suppression of reperfusion VT by PC was prevented by naloxone (VT incidence, 70%). The incidence of reperfusion VF was similarly low in the naloxone-treated rats in both nonpreconditioned and preconditioned groups (20% vs. 0). In the second series of experiments, the effect of 5-HD on repetitive PC was assessed. Repetitive PC was performed with three cycles of 2-min ischemia/5-min reperfusion, which totally abolished reperfusion VT and VF. This antiarrhythmic effect of repetitive PC was not inhibited by 5-HD. These results suggest that Katp channels and opioid receptors may be partly involved in suppression of reperfusion arrhythmias, although their roles may be compensated for by other antiarrhythmic mechanisms in repetitive PC. In contrast with PC against infarction, PKC is unlikely to play a major role in PC against reperfusion arrhythmias in the rat.