The effects of diquat metabolism on the protein thiol (PrSH) status of bis-chloronitrosourea-pretreated hepatocytes have been studied. Using a conventional, dithionitrobenzene-based assay for free PrSH in trichloroacetic acid-precipitated protein, control levels of PrSHs (83 +/- 6 nmol/mg protein) were unaltered during the initial 60 min of incubation of the cells with 1 mM diquat. However, using a radiochemical method for the determination of glutathionylation of PrSH [Grimm et al., Biochim Biophys Acta 844: 50-54, 1985], in which the hepatocytes were prepared from diethylmaleate-pretreated animals and reloaded with reduced glutathione (GSH) in the presence of [35S]methionine and cycloheximide, oxidation of hepatocellular PrSH by stimulated S-thiolation with GSH could be demonstrated. The S-glutathionylation of the protein was maximal after 30 min of treatment of the cells and preceded the onset of membrane leakage. However, the quantity of GSH mixed disulfide formed was limited to a maximum of 1.4 +/- 0.4 nmol GSH/mg protein, indicating the oxidation of only 2% of the total hepatocellular PrSH by S-thiolation. This percentage depletion is below the working variability of the colourimetric PrSH assay utilized and indicates strongly the use of the S-thiolation assay in the study of the possible effects of other redox-cycling cytotoxins on cellular PrSH status, as these may not be evident with conventional spectrophotometric techniques. The analysis of the cellular protein from diquat-treated cells by SDS-PAGE and autoradiography indicated the S-glutathionylation of a variety of cellular proteins, including species with molecular masses 17, 24, 26, 30, 40, 43 and 46 kDa. Although the identities of these species are uncertain (the 30-kDa protein may be equivalent to carbonic anhydrase as reported by Rokutan et al., Biochemistry 179: 233-239, 1989), it may be that oxidative modification of these proteins by stimulated S-glutathionylation may be an important early event in the mechanism of the hepatotoxicity of diquat.