Both tyrosine kinase (TK) and protein kinase C (PKC) inhibitors have been shown individually to completely abolish the cardioprotective effects of ischemic preconditioning (IPC) in rabbits; however, blockade of both enzymes is necessary to totally abolish IPC in pigs. Recently, we have shown that TK inhibition partially attenuates the cardioprotective effect of IPC in intact rat hearts. Therefore, the present study was designed to test the hypothesis that inhibition of both TK and PKC is necessary to completely abolish IPC in the intact rat and that this effect is dependent on the intensity of the preconditioning stimulus. All animals were subjected to 30 min of coronary artery occlusion and 2 h of reperfusion. In series 1, multiple-cycle-induced IPC was produced via three 5-min occlusions interspersed with 5 min of reperfusion (3 x 5 IPC). Genistein (5 mg/kg), a TK inhibitor infused 30 min before IPC, and chelerythrine chloride (5 mg/kg), a PKC inhibitor infused 5 min before the prolonged ischemic insult, were administered alone or in combination in the absence or presence of 3 x 5 IPC. 3 x 5 IPC produced a marked reduction in infarct size as a percentage of area at risk compared with control (8.0 +/- 0.8 vs. 56.1 +/- 0.8%). The effects of 3 x 5 IPC were partially blocked by pretreatment with genistein (34.0 +/- 2.0%) or chelerythrine (26.4 +/- 2.8%) alone; however, combined administration of genistein and chelerythrine completely abolished the effects of 3 x 5 IPC (50.7 +/- 3.6%). In series 2, single-cycle IPC was elicited by one 5-min occlusion followed by 10 min of reperfusion (1 x 5 IPC). Compared with control, 1 x 5 IPC also significantly reduced infarct size (15.4 +/- 3.0%). Genistein or chelerythrine administered alone completely abolished 1 x 5 IPC-induced cardioprotection. These results suggest that the efficacy of TK and PKC inhibition to block IPC depends on the intensity of the preconditioning stimulus and that these kinases may work through parallel pathways.