The oxidative inactivation of rabbit skeletal muscle Ca2+-ATPase in sarcoplasmic reticulum (SR) vesicles by peroxynitrite (ONOO−) was investigated. The exposure of SR vesicles (10 mg/ml protein) to low peroxynitrite concentrations (≤0.2 mM) resulted in a decrease of Ca2+-ATPase activity primarily through oxidation of sulf-hydryl groups. Most of this deactivation (ca. 70%) could be chemically reversed by subsequent reduction of the enzyme with either dithiothreitol (DTT) or sodium borohydride (NaBH4), indicating that free cysteine groups were oxidized to disulfides. The initial presence of 5 mM glutathione failed to protect the SR Ca2+-ATPase activity. However, as long as peroxynitrite concentrations were kept≤0.45 mM, the efficacy of DTT to reverse Ca2+-ATPase inactivation was enhanced for re action mixtures which initially contained 5 mM gluta thione. At least part of the disulfides were formed intermolecularly since gel electrophoresis revealed protein aggregation which could be reduced under reducing conditions. The application of higher Peroxnitrite concentrations (≥0.45 mM) resulted in Ca2+-ATPase in activation which could not be restored by exposure of the modified protein to reducing agents. On the other hand, treatment of modified protein with NaBH4recovered all SR protein thiols. This result indicates that possibly the oxidation of other amino acids contributes to enzyme inactivation, corroborated by amino acid analysis which revealed some additional targets for peroxynitrite or peroxynitrite-induced processes such as Met, Lys, Phe, Thr, Ser, Leu and Tyr. Tyr oxidation was confirmed by a significant lower sensitivity of oxidized SR proteins to the Lowry assay. However, neither bityrosine nor nitrotyrosine were formed in significant yields, as monitored by fluorescence spectroscopy and immunodetection, respectively. The Ca2+-ATPase of SR is involved in cellular Ca2+-homeostasis. Thus, peroxynitrite mediated oxidation of the Ca2+-ATPase might significantly contribute to the loss of Ca2+-homeostasis observed under biological conditions of oxidative stress.