The enzyme steroid 5 alpha-reductase catalyzes the production of dihydroprogesterone and dihydrotestosterone, which were recently recognized as neurosteroids in the brain with variably potential neuroactivity. The present study reports for the first time detailed localization of 5 alpha-reductase type I in the rat main olfactory bulb. The occurrence of 5 alpha-reductase in the olfactory bulb was detected by reverse transcription-polymerase chain reaction and Western blotting analyses. In addition, the enzyme activity was also detected by thin layer chromatography. Immunocytochemistry showed that 5 alpha-reductase immunoreactive cells of variable intensity were present in all layers of the olfactory bulb. Multiple immuno-labeling revealed that 5 alpha-reductase was mainly localized in glial cells, namely, in S-100 beta- and glial fibrillary acidic protein-immunoreactive astrocytes, 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase)-immunoreactive oligodendrocytes, and in S-100 beta- and neuropeptide-Y-immunoreactive olfactory ensheathing cells, whereas the bulbar neurons exhibited little immunoreactivity. Quantitative analysis revealed that the number of 5 alpha-reductase-immunoreactive cells was greatest in the olfactory nerve layer. The most intense 5 alpha-reductase-immunoreactivity was found in the olfactory ensheathing cells, and next in the CNPase-immunoreactive cells. The 5 alpha-reductase in the olfactory bulb was expressed constantly throughout different ages and sexes and in neutered and hypophysectomized rats. Thus, 5 alpha-reductase may contribute via 5 alpha-reduced metabolites to the formation and maintenance of olfactory inputs and outputs, which were closely associated with the olfactory ensheathing cells and the oligodendrocytes, respectively.