1. The addition of n-butyl nitrite (BN) to isolated rat hepatocytes caused rapid S-nitrosyl glutathione (GSNO) formation, then a concomitant decrease in protein thiols, followed by a marked ATP depletion. Cytotoxic concentrations of BN also caused lipid peroxidation after a long lag period but before cytotoxicity ensued. 2. Prior glutathione (GSH) depletion protected hepatocytes against the BN-induced decrease in protein thiols, ATP depletion, lipid peroxidation and cytotoxicity. Thus cytotoxic effects were thought to be mediated via GSNO formed by reaction of BN with GSH, a reaction catalysed by the cytosolic fraction. 3. Cytotoxicity and lipid peroxidation, but not depletion of GSH, protein thiols or ATP, could be averted by the subsequent addition of antioxidants or the iron chelator, desferoxamine. 4. Addition of the thiol reductant, dithiothreitol to BN-treated hepatocytes restored GSH and protein thiols and also prevented cytotoxicity.