The sarcoplasmic reticulum (SR) calcium ATPase carries out active Ca 2+ pumping at the expense of ATP hydrolysis. We have previously described the inhibition of SR ATPase by oxidative stress induced by the Fenton reaction (Fe 2+ + H 2O 2 → HO + HO − + Fe 3+). Inhibition was not related to peroxidation of the SR membrane nor to oxidation of ATPase thiols, and involved fragmentation of the ATPase polypeptide chain. The present study aims at further characterizing the mechanism of inhibition of the Ca 2+-ATPase by oxygen reactive species at Fe 2+ concentrations possibly found in pathological conditions of iron overload. ATP hydrolysis by SR vesicles was inhibited in a dose-dependent manner by micromolar concentrations of Fe 2+, H 2O 2, and ascorbate. Measuring the rate constants of inactivation ( k inact ) at different Fe 2+ concentrations in the presence of saturating concentrations of H 2O 2 and ascorbate (100 μM each) revealed a saturation profile with half-maximal inactivation rate at ca. 2 μM Fe 2+. Inhibition was not affected by addition of 200 μM Ca 2+ to the medium, indicating that it was not related to iron binding to the high affinity Ca 2+ binding sites in the ATPase. Furthermore, inhibition was not prevented by the water-soluble hydroxyl radical scavengers mannitol or dimethylsulfoxide, nor by butylated hydroxytoluene (a lipid peroxidation blocker) or dithiothreitol (DTT). However, when Cu 2+ was used instead of Fe 2+ in the Fenton reaction, ATPase inhibition could be prevented by DTT. We propose that functional impairment of the Ca 2+-pump may be related to oxidative protein fragmentation mediated by site-specific Fe 2+ binding at submicromolar or low micromolar concentrations, which may occur in pathological conditions of iron overload.