Protein S-thiolation was characterized in cultured hepatocytes treated with t-butyl hydroperoxide, menadione and phorbol diester-stimulated neutrophils. A similar group of soluble proteins was quickly S-thiolated in oxidant-treated hepatocytes and glutathione contributed at least 90% of the total protein-bound thiols. In hepatocytes treated with 0.5 mM t-butyl hydroperoxide, 0.2 mM menadione or 10[superscript]6 neutrophils, the maximum amount of protein-bound thiols was shown to be approximately 10%, 24% and 8% of cellular glutathione respectively bound to proteins. The most abundant S-thiolated 30 kDa protein in hepatocytes was identified as carbonic anhydrase III in all cases. In oxidant-treated cells carbonic anhydrase III contributed 30% of the total amount of protein-bound thiols. Two major S-thiolated proteins, (46 and 62 kDa) were observed in untreated hepatocytes and S-thiolation of these two proteins was not increased by the addition of oxidants to cells. The amount of protein S-thiolation was not directly correlated with the formation of glutathione disulfide in t-butyl hydroperoxide and menadione-treated hepatocytes. When hepatocytes were treated with stimulated neutrophils, there was a substantial amount of protein S-thiolation in hepatocytes with no increase in the concentration of glutathione disulfide in hepatocytes. Two postulated mechanisms contributed to protein S-thiolation in hepatocytes during oxidative stress;Protein S-thiolation occurred in phorbol-diester stimulated neutrophils. Glutathione contributed at least 85% of the total protein-bound thiol and homocysteine or cysteine contributed modestly to protein S-thiolation. After stimulation for 30 min approximately 10% of the cellular glutathione was bound to proteins. There was no increase of glutathione disulfide during stimulation suggesting that S-thiolation of proteins does not occur by thiol/disulfide exchange in neutrophils. A major S-thiolated 42 kDa protein was identified as actin in neutrophils. F-actin was preferentially S-thiolated. S-thiolation of actin may affect the function of NADPH-oxidase in neutrophils.