Previous studies in our laboratory demonstrated the alteration in the physical state of synaptosomal membrane lipids and proteins in ischemia/reperfusion injury using selective spin labels and electron paramagnetic resonance spectroscopy [Hall et al. (1995) Neuroscience 61, 84-89]. Since many investigations have provided evidence for free radical generation during ischemia/reperfusion injury, we investigated whether a free radical scavenger would prevent the membrane damage, in gerbils. Further, experiments to determine if a secondary effect of polyamine generation at 14 h reperfusion could be blocked by this free radical scavenger or by an inhibitor of ornithine decarboxylase were also carried out. The alterations in synaptosomal membrane integrity observed during ischemia/reperfusion injury were selectively neutralized by treatment with the free radical spin trap N-tert-butyl-alpha-phenylnitrone or an inhibitor of ornithine decarboxylase, difluoromethylornithine. Administration of N-tert-butyl-alpha-phenylnitrone prior to ischemia totally abrogated both lipid and protein alterations observed at 1 and 14 h reperfusion. Pretreatment with difluoromethylornithine neutralized only the 14 h change in lipid label motion. Treatment with N-tert-butyl-alpha-phenylnitrone at 6 h post ischemia showed only a slight attenuation of the 14 h lipid effect and no change in the protein effect. Difluoromethylornithine treatment at 6 h post ischemia negated the 14 h ischemia/reperfusion injury-induced lipid effect and had no effect on the protein change. These data support previous suggestions that free radicals and polyamines play a critical role in neuronal damage and cell loss following ischemia/reperfusion injury and that the polyamine effect is dependent upon free radical generation during ischemia/reperfusion injury.