Evidence that partially oxidized piperidine derivatives such as the Parkinsonian inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are biotransformed in a reaction catalyzed by monoamine oxidase B (MAO-B) to neurotoxic pyridinium metabolites led to studies resulting in the identification of the haloperidol-derived pyridinium metabolite in the urine of drug-treated rats. The present in vitro studies examine the metabolic pathway governing this overall four-electron oxidation. Although haloperidol and its 1,2,3,6-tetrahydropyridine dehydration product were not substrates for purified bovine liver MAO-B, both compounds were biotransformed to the pyridinium product by rat liver microsomal preparations. The dependence on NADPH and the inhibition by SKF-525A argue that one or more liver cytochrome P-450 isozymes may catalyze this transformation. Attempts to detect possible metabolic intermediates were not successful. Chemical model studies, however, suggest that the expected intermediary amino enol and dihydropyridinium species may be too unstable to isolate. The possible significance of this pathway with respect to haloperidol-induced central nervous system dysfunction is considered.