Sarcoplasmic reticulum (SR) microsomes were oxidized by exposure to peroxydisulfate, hydrogen peroxide, or iron/ascorbate or by extended storage. The decline in Ca2+-ATPase activity, Ca2+ transport, and the increase in Ca2+ permeability which occurred under these conditions did not appear to result from lipid oxidation because these functional changes were not correlated with the amount of thiobarbituric acid-reactive lipid. Consistent with this interpretation, lipid antioxidants did not prevent the decline in SR function. This suggests that inhibition was independent of lipid oxidation. Instead, oxidation directly inhibited the Ca2+-ATPase. The decline in enzyme activity may be due to oxidation of SH groups, as suggested by the ability of reducing agents to prevent inhibition, the decline in sulfhydryl content of oxidized SR, and the ability of sulfhydryl-binding agents to inhibit Ca2+-ATPase. Inhibition was not primarily due to crosslinking of the Ca2+-ATPase, because sodium dodecyl sulfate-polyacrylamide gels of normal and oxidized SR showed that the area of the Ca2+-ATPase band was not correlated with the Ca2+-ATPase activity. Inhibition of the Ca2+-ATPase by oxidative stress is relevant to models of cellular dysfunction in which toxicity is caused by a rise in intracellular calcium.