We have reported that strenuous physical exercise causes a decrease in protein-bound sulfhydryl groups (p-SHs), such as albumin cysteine residues, in human plasma (Inayama et al., 1996, Life Sci., 59, 573-578). We further investigated the fate of plasma protein thiols after moderate exercise. Six untrained healthy female volunteers ran for 30-min at the individual ventilatory threshold. We observed an increase in protein cysteine mixed disulfides (p-S-Cys) after running, as evidenced by reducing plasma proteins with dithiothreitol to detect the increase of cysteine, along with the concomitant decrease in p-SHs in plasma. However, plasma protein-bound glutathione (GSH) and S-nitroso-protein were undetectable before and after exercise. Test tube experiments suggest that p-S-Cys are probably formed by the hydrolysis of protein GSH mixed disulfides by γ-glutamyltranspeptidase and peptidase, and/or by the oxidative addition of p-SHs to cystine.