The enzymatic conversion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to 1-methyl-4-phenylpyridinium ion by monoamine oxidase-B is an essential step mediating the dopaminergic neurotoxicity. Since monoamine oxidase-B is located primarily in serotonergic neurons and astrocytes, the production of 1-methyl-4-phenylpyridinium ion is thought to be extra-dopaminergic. This study provides evidence in support of this conclusion. Pretreating mice with fluoxetine (a serotonergic uptake inhibitor) before the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine attenuated the dopaminergic neurotoxicity. This was not the result of a nonspecific inhibition of dopaminergic uptake, as fluoxetine pretreatment did not attenuate the dopaminergic neurotoxicity resulting from the intrastriatal administration of the 1-methyl-4-phenylpyridinium ion. Further localization of the primary site of 1-methyl-4-phenylpyridinium ion production as being astrocytes was provided by the failure of 5,7-dihydroxytryptamine-induced serotonergic lesions to attenuate the neurotoxicity produced by administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, whereas, fluoxetine pretreatment in similarly lesioned subjects, continued to attenuate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurotoxicity. These results are consistent with the hypothesis that astrocytes are a principle site of 1-methyl-4-phenylpyridinium ion production.