Monolayer cultures of pig hepatocytes were used to investigate the role of the liver in the biotransformation of the veterinary drug dimetridazole (1,2-dimethyl-5-nitroimidazole). 14C-Labeled dimetridazole (DMZ) was primarily hydroxylated to 1-methyl-2-hydroxymethyl-5-nitroimidazole (up to 90%) and to a minor extent N-demethylated to 2-methyl-(4,5)-nitroimidazole (6-10%). Prolonged incubation with the parent drug but also the 2-hydroxymethyl metabolite resulted in the formation of two other minor metabolites. The major one of these was identified as the glucuronide of the 2-hydroxymethyl metabolite, based on its molecular mass and the succesful hydrolysis with β-glucuronidase. The second showed a molecular mass of 144 and is hypothesized to be 2-hydroxymethyl-5-nitroimidazole. No evidence was obtained for the formation of a cysteine or glutathione conjugate involved in the detoxification of reactive intermediates. In addition to free metabolites, there was a time-related formation of protein-bound metabolites up to a maximum of 30 pmol/mg of protein after exposure to 50 μM DMZ for 48 h. In general, these metabolites accounted for 0.06-0.15% of the metabolized DMZ. Unextractable metabolites were also observed after incubation of cells with the 2-hydroxymethyl and 1-desmethyl metabolites. It is concluded that the 1-desmethyl and, in particular, the 2-hydroxymethyl metabolite are the major metabolites formed by pig hepatocytes. These compounds are thus far the only metabolites identified in vivo, but together with the parent compound, they accounted for only a small part (<5%) of the excreted drug. Therefore, the site of formation of the majority of the unknown in vivo metabolites may be extrahepatic, and complimentary models are needed to investigate this hypothesis.