In order to better understand the first steps leading to drug-induced immunoallergic hepatitis, we studied the target of anti-LKM2 autoantibodies appearing in tienilic acid-induced hepatitis, and the target of tienilic acid-reactive metabolites. It was identified as cytochrome P450 2C9, (P450 2C9): indeed, anti-LKM2 specifically recognized P450 2C9, but none of the other P450s tested (including other 2C subfamily members, 2C8 and 2C18). Tienilic acid-reactive metabolite(s) specifically bound to P450 2C9, and experiments with yeast expressing active isolated P450s showed that P450 2C9 was responsible for tienilic acid-reactive metabolite(s) production. Results of qualitative and quantitative covalent binding of tienilic acid metabolite(s) to human liver microsomes were then compared to those obtained with two drugs leading to direct toxic hepatitis, namely, acetaminophen and chloroform. Kinetic constants (Km and Vmax) were measured, and the covalent binding profile of the metabolites to human liver microsomal proteins was studied. Tienilic acid had both the lowest Km and the highest covalent binding rate at pharmacological doses. For acetaminophen and chloroform, several microsomal proteins were covalently bound, while covalent binding was highly specific for tienilic acid and dihydralazine, another drug leading to immunoallergic hepatitis. Although low numbers of drugs were tested, these results led us to think that there may exist a relationship between the specificity of covalent binding and the type of hepatotoxicity.