The hepatocyte metabolic pathways involved in the detoxification of cyanide by cysteine have been investigated in vitro using hepatocytes isolated from Sprague-Dawley rats. Cyanide toxicity towards isolated hepatocytes could be prevented by the addition of L- or D-cysteine, cystine, or the cysteine metabolites thiosulfate and mercaptopyruvate, which markedly increased thiocyanate formation. Prior depletion of hepatocyte GSH markedly increased thiosulfate formation from L- or D-cysteine without affecting thiocyanate formation from L- or D-cysteine. This suggested that the major metabolic pathway for thiocyanate formation did not involve thiosulfate. Mercaptopyruvate was a more likely metabolic intermediate, as thiocyanate formation from L-cysteine but not thiosulfate was inhibited markedly by aminooxyacetate, a cysteine aminotransferase inhibitor, and propargylglycine, a gamma-cystathionase inhibitor. Furthermore, propargylglycine prevented L-cysteine cytoprotection against cyanide toxicity. Thiocyanate formation from D-cysteine likely also involved mercaptopyruvate, as thiocyanate formation from D-cysteine but not L-cysteine was inhibited by benzoate, an inhibitor of D-amino acid oxidase. Furthermore, benzoate prevented D-cysteine cytoprotection against cyanide toxicity. Cystine may also be an intermediate, as hepatocyte thiocyanate formation from added L-cysteine was inhibited when L-cysteine autoxidation was prevented with the copper chelator bathocuproine disulfonate. Furthermore, thiocyanate formation by rat liver homogenates with L-cystine was far more rapid than that with L-cysteine. Hepatocyte thiocyanate metabolic intermediates of beta-mercaptopyruvate and thiocystine were proposed for L-cysteine, and beta-mercaptopyruvate was proposed for D-cysteine.