Recent interest in the neurotoxicity of haloperidol is based on its oxidation in rodents to the pyridinium derivative, HPP+, a structural analog of the neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). Recently, we reported that HPP+ and a newly identified reduced pyridinium, RHPP+, were present in blood and urine of haloperidol-treated schizophrenics and that the concentrations of RHPP+ exceeded those of HPP+. In this study, we examined pathways for formation of RHPP+ in subcellular fractions of human liver (n = 5) and brain (basal ganglia; n = 5). The major pathway was reduction of HPP+ (20 microM) to RHPP+ in cytosol (0.17-0.39 and 0.03-0.07 microM RHPP+/g cytosolic protein per h in liver and brain, respectively). The reactions were inhibited significantly by menadione and in brain also by daunorubicin. The inhibition profile, cytosolic location and strict NADPH dependence suggest that the enzymes involved are ketone reductases. A second pathway was oxidation of reduced haloperidol (50 microM), a major metabolite of haloperidol in blood and brain, to RHPP+. In liver microsomes, 0.17-0.63 mumol RHPP+ was formed /g microsomal protein per h. A potent inhibitor of the pathway was ketoconazole (IC50, 0.8 microM), which suggests that P-450 3A isozymes could be involved. In brain mitochondria but not microsomes, reduced haloperidol (120 microM) was oxidised to RHPP+ at a small but significant rate (0.005-0.020 mumol RHPP+/g mitochondrial protein per h) which was not attenuated by SKF 525A, quinidine, ketoconazole, or monoamine oxidase inhibitors. Further studies are warranted to establish the biological importance of these metabolites in vivo.