Recent studies demonstrated that brief period of Ca2+ depletion and repletion (Ca2+ preconditioning, CPC) has strong protective effects against ischemia in a rat heart. CPC and classic preconditioning (IPC) were compared in relation with infarct size and protein kinase C (PKC) isozymes. Isolated Langendorff-perfused rabbit hearts were subjected to 45-min ischemia (Isc) followed by 120-min reperfusion (R) with or without IPC, induced by 5-min Isc and 10-min R. In the CPC hearts, 5-min Ca2+ depletion and 10-min repletion (CPC) were given before 45-min Isc, with or without concurrent PKC inhibition (calphostin C, 200 nmol/L). IPC enhanced recovery of LV function, while CPC did not. Infarct size was significantly reduced by both CPC and IPC (p < 0.05 vs. ischemic control). Membrane PKC was significantly increased from 2.53 ± 0.07 (baseline, nmol/g tissue) to 3.11±0.07, 3.34 ± 0.11, 3.15 ± 0.09, and 3.06 ± 0.08 by IPC, IPC and 45-min Isc, CPC and 45-min Isc, respectively (p < 0.01). Immunoblots of membrane PKC were increased by IPC, IPC and 45-min Isc, and CPC. These effects were abolished by PKC inhibition. Thus, activation of PKC may have trigger role in the mechanism of cardioprotective effect by CPC.