Inhibition of Na+/K(+)-ATPase by partially reduced oxygen metabolites has been suggested to be involved in ischemia-reperfusion injury to heart and other organs. Since various isoforms of the enzyme have different sensitivities to ouabain and several other inhibitors, we studied the effects of H2O2 and the hydroxyl radical on enzyme activity and phosphoenzyme formation in Na+/K(+)-ATPase preparations with known alpha-subunit isoform composition in order to assess the oxidant sensitivities of the isoforms. Rat axolemma enzyme (alpha 2 and alpha 3) which has higher sensitivity than the rat kidney enzyme (alpha 1) to ouabain also showed higher oxidant sensitivity than the kidney enzyme. No significant difference between the oxidant sensitivities of the alpha 2 and alpha 3 of the axolemma was noted. In the ferret heart enzyme (alpha 1 and alpha 3), we confirmed that alpha 3 has higher ouabain sensitivity than alpha 1, and we established that alpha 3 also has higher oxidant sensitivity than alpha 1. The rat kidney enzyme (alpha 1) and the canine kidney enzyme (a variant of alpha 1 with much higher ouabain sensitivity than the rat kidney enzyme) exhibited similar oxidant sensitivities. The findings suggest that (a) oxidant sensitivity is related to structural features that distinguish alpha 1 from alpha 2 and alpha 3, rather than to features that control ouabain sensitivity; and (b) different isoform compositions of the various tissues may contribute to their relative susceptibilities to oxidant stress.