The mechanism of propranolol-inhibited sarcolemmal membrane lipid peroxidation was investigated by electron spin resonance spin-trapping technique using 5,5-dimethyl- 1-pyrroline-N-oxide (DMPO) and 2-methyl-2-nitrosopropane (MNP). Highly purified canine myocytic sarcolemma were peroxidized by a superoxidc-driven (from dihydroxyfumarate) and Fe3+-catalyzed free radical-generating system. Hydroxyl radicals (-OH), identified by electron spin resonance signals as DMPO-OH adducts, were generated in the aqueous phase. Propranolol up to 500 μM did not effectively reduce the intensity of the DMPO-OH adducts. When the sarcolemma were incubated with MNP before the addition of free radicals, MNP adducts characteristic of carbon-centered radicals were produced. Pretreatment of the membranes with propranolol (3-100 μM) decreased the intensity of the MNP adducts in a log concentrationdependent manner; the EC50is about 7 μM. D- and L-propranolol were found equally effective. When protein-depleted sarcolemmal lipids were similarly incubated with MNP and the free radical system, identical MNP adducts were observed; this finding suggests that the adducts were lipid-derived products arising from lipid peroxidation. Furthermore, their formation was also inhibited by propranolol pretreatment. Since propranolol is not an effective scavenger of oxygen radicals in the aqueous phase, the data suggest that the antiperoxidative effect of propranolol is due to its lipophilic interaction with the membrane and thus subsequent interruption of the free radical chain reactions.