Hokkaido brown bears (Ursus arctos yesoensis) are seasonal breeders and the profile of their serum testosterone concentrations undergoes annual changes. However, precise sites of steroidogenesis in the bear testis have not been identified. Therefore, our objective was to localize steroidogenic enzymes by immunocytochemistry using polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. The steroidogenic enzymes localized were cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17 alpha-hydroxylase cytochrome P450 (P450c17), and aromatase cytochrome P450 (P450arom) as biosynthetic sites of pregnenolone, progesterone or androstenedione, androgens, and estrogens, respectively. Testes were collected from three adult bears prior to the mating season (April, Hokkaido, Japan) and prepared for immunostaining with primary antibodies, followed by an avidin-biotin-peroxidase method. P450scc was localized in Leydig cells and spermatids. 3 beta-HSD was found only in Leydig cells. P450c17 was identified in Leydig cells and spermatids. Finally, P450arom was found in Leydig cells and in spermatids that stained very intensely. Therefore, Leydig cells appear to be a site of progestin (both delta 4 and delta 5 C21 steroids), androgen, and estrogen production, whereas spermatids appear to be a site of pregnenolone, androgen, and estrogen production. Immunolocalization of steroidogenic enzymes suggests that steroidogenesis may occur not only in Leydig cells, but also in spermatids of the Hokkaido brown bear testis prior to the mating season. We also suggest that Leydig cells and spermatids are the predominant sites of androgen and estrogen synthesis, respectively, in the Hokkaido brown bear testis.