Protein kinase C (PKC) inhibitors, chelerythrine (Chel, 0.6 mg) and polymyxin B (Poly B, 1.0 mg), and PKC activators, phorbol 12-myristate 13-acetate (PMA, 0.05 mg) and 1-oleoyl-2-acetyl glycerol (OAG, 0.1 mg), were used as probes to investigate the role of PKC in mediation of ischemic preconditioning (IPC) of noncontracting pig latissimus dorsi (LD) muscles against infarction in vivo. These drugs were delivered to each LD muscle flap (8 × 12 cm) by 10 min of local intra-arterial infusion. It was observed that LD muscle flaps sustained 43 ± 5% infarction when subjected to 4 h of global ischemia and 24 h of reperfusion. IPC with three cycles of 10 min ischemia-reperfusion reduced muscle infarction to 25 ± 3% (P < 0.05). This anti-infarction effect of IPC was blocked by Chel (42 ± 7%) and Poly B (37 ± 2%) and mimicked by PMA (19 ± 10%) and OAG (14 ± 5%) treatments (P < 0.05), given 10 min before 4 h of ischemia. In addition, the ATP-sensitive K⁺ (K_ATP) channel antagonist sodium 5-hydroxydecanoate attenuated (P < 0.05) the anti-infarction effect of IPC (37 ± 2%), PMA (44 ± 17%), and OAG (46 ± 9%). IPC, OAG, and Chel treatment alone did not affect mean arterial blood pressure or muscle blood flow assessed by 15-µm radioactive microspheres. Western blot analysis of muscle biopsies obtained before (baseline) and after IPC demonstrated seven cytosol-associated isoforms, with nPKC alone demonstrating progressive cytosol-to-membrane translocation within 10 min after the final ischemia period of IPC. Using differential fractionation, it was observed that nPKC translocated to a membrane compartment other than the sarcolemma and/or sarcoplasmic reticulum. Furthermore, IPC and preischemic OAG but not postischemic OAG treatment reduced (P < 0.05) muscle myeloperoxidase activity compared with time-matched ischemic controls during 16 h of reperfusion after 4 h of ischemia. Taken together, these observations indicate that PKC plays a central role in the anti-infarction effect of IPC in pig LD muscles, most likely through a PKC-K_ATP channel-linked signal-transduction pathway.