We examined the roles of nitric oxide and protein kinase C (PKC) in ACh-produced protection of cultured cardiomyocytes during simulated ischemia and reoxygenation. Cell viability was quantified using propidium iodide in chick embryonic ventricular myocytes. O₂ radicals were quantified using 2',7'-dichlorofluorescin diacetate. After a 10-min infusion of ACh (0.5 or 1 mM) and a 10-min drug-free period, we simulated ischemia for 1 h and reoxygenation for 3 h. ACh reduced cardiocyte death [32 ± 4%; n = 6 and 23 ± 4%; n = 7 (P < 0.05)] and attenuated oxidant stress during ischemia and reoxygenation in a concentration-dependent manner compared with controls (47 ± 4%; n = 8; P < 0.05). The increase in O₂ radicals before simulated ischemia [357 ± 49; n = 4 and 528 ± 52; n = 8 vs. 211 ± 34; n = 8; P < 0.05 (arbitrary units)] was abolished by the specific nitric oxide synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) and was markedly attenuated by N^G-monomethyl-L-arginine (L-NMMA). L-NAME or L-NMMA blocked the protective effects of ACh, which selectively increased PKC- isoform activity in the particulate fraction. The PKC inhibitor Gö-6976 had no effect on O₂ radical production before simulated ischemia but it abolished the protection; therefore nitric oxide is a large component of ACh-generated O₂ radicals. Nitric oxide and O₂ radicals activate the PKC- isoform by which ACh protects against injury.