Type I 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) is mainly involved in the reductive transformation of estrone to estradiol. Such a conversion is known to occur in mammalian brain. In order to determine the brain areas and the nerve cell types containing this enzyme, we have proceeded to its immunocytochemical localization in the adult rat brain. Immunoblot analysis showed that the antibodies used could specifically bind to one brain protein band corresponding to purified 17 beta-HSD. Immunolabelled cells were found in high concentration in the hypothalamus, thalamus, hippocampus, cerebral cortex, caudate putamen and pineal gland. At the light microscopic level, 17 beta-HSD immunoreactive material appeared to be present only in glial and ependymal cells, including tanycytes. Double staining procedures showed that the 17 beta-HSD nerve cells also contained glial fibrillary acidic protein (GFAP), a specific marker for glial cells. Immunoelectron microscopic studies demonstrated that immunoreactive material was diffusely distributed throughout the cytoplasm of glial and ependymal cells, thus confirming the association of 17 beta-HSD immunoreactivity with nonneuronal cells. These data suggest that glial cells play an important role in the conversion of a weak estrogen, estrone, to a more potent estrogen, estradiol.