A certain number of case reports of adverse hepatic reactions to diclofenac are known, suggesting that diclofenac-associated hepatitis may be more common than previously recognized. In order to discriminate among possible molecular mechanisms of toxicity, the following were investigated: (a) cytotoxicity of diclofenac on metabolizing (rat hepatocytes) and non-metabolizing hepatic cells (HepG2, FaO); (b) changes in calcium homoeostasis, glutathione (GSH), lipid peroxidation and ATP levels, and (c) diclofenac metabolism in relation to cytotoxicity. The results indicate that toxicity is associated with the oxidative metabolism of the drug, and correlated with the formation of a minor oxidation metabolite. Inhibitors of diclofenac metabolism concomitantly reduced the toxicity of the drug. Hepatocyte injury was preceded by a decrease in ATP levels. No oxidative stress (no changes in GSH, no lipid peroxidation) could be demonstrated at this early stage. Cytotoxicity was prevented when cells were incubated with fructose, suggesting that the inability of mitochondria to produce ATP is the probable cause of diclofenac hepatotoxicity.