Catechol-O-methyltransferase (COMT)(EC 2.1.1.6) was localized using fluorescence immunohistochemistry in rat liver and kidney and in rat, chinchilla, and bovine brains. In the brain, specific fluorescence was visable only in non-neuronal cellular elements in all three species. Ventricular ependymal cells and cells of the choroid plexuses exhibited the greatest intensity of immunofluorescence. Glial immunofluorescence appeared prominently in large myelinated fiber tracts. Interfascicular and perineuronal satellite oligodendrocytes as well as fibrous astrocytes were immunoreactive, though myelin itself did not exhibit a positive reaction. Bergmann glial cells in the cerebellum also stained brightly for COMT. Although this study indicates that the predominant localization of COMT is non-neuronal, it is important to note that the presence of small quantities of this enzyme in neurons cannot be excluded. The patterns of localization observed in the non-neuronal elements suggest that this enzyme may function as a barrier to free diffusion of catechol compounds within the central nervous system.