Sodium nitroprusside (SNP) cytotoxicity towards rat hepatocytes was accompanied by peroxynitrite formation, lipid peroxidation, inhibition of glycolysis, cyanide (CN) release, partial inhibition of hepatocyte respiration, and ATP depletion. Antioxidants and desferoxamine prevented both cytotoxicity and lipid peroxidation induced by SNP. The CN antidote thiosulfate or the CN trapping agents dihydroxyacetone and glyceraldehyde increased SNP metabolism, SNP-induced peroxynitrite formation, cytotoxicity, and lipid peroxidation. On the other hand, addition of non-toxic concentrations of CN to hepatocytes prevented SNP metabolism and SNP-induced lipid peroxidation and cytotoxicity. SNP depleted hepatocyte GSH immediately upon addition, and GSH-depleted hepatocytes were more susceptible to SNP. The results of this study suggest that nitric oxide rather than CN mediates SNP cytotoxicity in isolated cells.