The effects of three cysteine pro-drugs on the hepatotoxicity and biotransformation of acetaminophen were examined to evaluate the factors responsible for antidotal effectiveness. N-Acetyl-L-cysteine, L-2-oxothiazolidine-4-carboxylate or the L- or D-isomers of 2-methylthiazolidine-4-carboxylate were administered to male mice immediately after a hepatotoxic dosage of acetaminophen (5.0 mmol/kg). In general the antidotal efficacies and potencies of the L-cysteine pro-drugs were similar; 5.0 mmol/kg prevented hepatotoxicity whereas moderate and no protection were observed after 1.65 and 0.55 mmol/kg, respectively. In contrast, the D-isomer of 2-methylthiazolidine-4-carboxylate was ineffective at all dosages. Both L-2-oxothiazolidine-4-carboxylate and L-2-methylthiazolidine-4-carboxylate enhanced blood acetaminophen elimination (28-31% decrease in half-life) whereas N-acetyl-L-cysteine and D-2-methylthiazolidine-4-carboxylate did not. The L-cysteine pro-drugs increased the urinary excretion of the cysteine and mercapturic acid conjugates of acetaminophen (34-119%) but did not alter excretion of acetaminophen-glucuronide or acetaminophen-sulfate. The D-cysteine pro-drug did not affect the urinary excretion of the acetaminophen metabolites examined. Biochemical analyses of the phase II pathway co-substrates, i.e., UDP-glucuronic acid, adenosine 3'-phospho-5'-phosphosulfate and glutathione, were performed on liver samples from mice treated with pro-drugs and/or acetaminophen. The pro-drugs exhibited their greatest effect on hepatic glutathione concentrations. Treatment with L-cysteine pro-drugs decreased the extent of depletion and/or increased the rate of repletion of hepatic glutathione levels after acetaminophen administration.(ABSTRACT TRUNCATED AT 250 WORDS)