The SR Ca-ATPase in skeletal muscle SR vesicles isolated from young adult (5 months) and aged (28 months) rats was analyzed for nitrotyrosine. Only the SERCA2a isoform contained significant amounts with approximately one and four nitrotyrosine residues per young and old Ca-ATPase, respectively. The in vitro exposure of SR vesicles of young rats to peroxynitrite yielded selective nitration of the SERCA2a Ca-ATPase even in the presence of excess SERCA1a. No nitration was observed during the exposure of SR vesicles to nitric oxide in the presence of O2. These data suggest the vivo presence of peroxynitrite in skeletal muscle. The greater nitrotyrosine content of SERCA2a from aged tissue implies an age-associated increase in susceptibility to oxidation by this species.