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.