Serum albumin is a mixture of mercaptalbumin (reduced form) and non-mercaptalbumin (oxidized form), i.e. a protein redox couple in serum. To investigate dynamic changes in the redox state of rat serum albumin (RSA), we developed a simple and sensitive high-performance liquid chromatographic (HPLC) system using an ion-exchange column with a linear gradient of ethanol concentration. Furthermore, we applied this HPLC system to examine dynamic changes in the redox state of RSA caused by severe oxidative stress such as exhaustive physical exercise. Using this system, we successfully separated RSA to rat mercaptalbumin (MAr) and rat non-mercaptalbumin (NAr), and also found the best conditions for the clear separation of RSA. In the experiments with exhaustive exercise, mean values for the MAr fraction in control and exercise groups were 76.2±1.8 and 69.0±3.5%, respectively. The MAr in the exercise group was significantly oxidized compared with that of the control group (P<0.01). These results suggested that RSA might act as one of the major scavengers in extracellular fluids under severe oxidative stress. © 2002 Elsevier Science B.V. All rights reserved.