Cu,Zn-Superoxide dismutase (SOD) was isolated from the liver of 3-, 12-, and 26-month-old Fisher 344 (F344) rats. Specific activity and metal content of the enzyme, purified by ion-exchange and size-exclusion chromatography, did not significantly change with age. Electrospray ionization-mass spectrometry and amino acid analysis of Cu,Zn-SOD apoprotein, further purified by reverse-phase HPLC, showed neither significant loss of amino acids nor accumulation of oxidized isoforms with age. When bovine Cu,Zn-SOD, oxidized with H₂O₂ in vitro, was added to rat liver homogenate, we reisolated circa 70% of the oxidized bovine Cu,Zn-SOD together with the rat isoform, showing that oxidized Cu,Zn-SOD can be recovered from tissue homogenate. Therefore, our data do not confirm an earlier hypothesis that oxidatively modified Cu,Zn-SOD protein accumulates in the liver of aged F344 rats.