The disulfide metabolites of thiono-sulfur drugs were found to be about 50 to 100 times more toxic to isolated rat hepatocytes than the corresponding parent drugs. The order of decreasing cytotoxicity for the disulfide metabolites was disulfiram greater than propylthiouracil disulfide greater than formamidine disulfide greater than phenylthiourea disulfide greater than thiobenzamide disulfide greater than cystamine. Depletion of intracellular GSH levels preceded cytotoxicity. GSH could be restored and cytotoxicity averted by adding the thiol reducing dithiothreitol. Depletion of GSH with diethylmaleate potentiated the toxicity of disulfides 3 to 4-fold confirming the protective role of GSH in disulfide toxicity. The toxicity of disulfiram was increased 4-fold in cells pretreated with ATP (0.8 mM) to effect a transient increase in cytosolic Ca2+ suggesting an impairment of Ca2+ homeostasis by the toxicant. Disulfiram (200 microM) rapidly depleted hepatocyte ATP levels within 15 minutes which suggests that ATP production is inhibited. The disulfide effectiveness at causing mitochondrial Ca2+ release was similar to their effectiveness at inducing hepatocyte cytotoxicity. These results suggest that hepatocyte toxicity is the result of oxidative inactivation of membrane protein thiols that regulate intracellular Ca2+ homeostasis.