The Japanese black bear (Ursus thibetanus japonicus), which is a subspecies of the Asiatic black bear (Ursus thibetanus) and inhabits Japan, is a seasonal breeder with the mating season in June to July. Male bears exhibit full spermatogenesis and high concentrations of testosterone in blood in the pre-mating season. Molecular mechanism of spermatogenesis, complicatedly regulated by factors such as sex steroid hormones and cytokines, is known to be different among species. Up to date, few report of steroidogenic enzymes detection at mRNA level in the testes of Carnivora has been published, in spite of many reports on immunolocalization of them. Especially, 17βHSD family has been recently analyzed for its structure, so its expression remains to be elucidated, although it is the key enzyme concerning testosterone synthesis in the testes. To investigate steroidogenesis in the testes of the Japanese black bear, we utilized the testes samples taken from 4 adult male bears in mating season. Partial mRNA sequence of five steroidogenic enzymes (P450scc, 3βHSD, P450c17, 17βHSD3 and P450arom) expressing in the Japanese black bear testes were determined and their expression was detected by means of in situ hybridization with 35S labeled 45-mer antisense oligonucleotide probes specific to each enzyme. Partial mRNA codes of P450scc, 3βHSD, P450c17, 17βHSD3, and P450arom i487bp, 229bp, 512bp, 719bp and 460bp, respectivelyj showed 91.1~95.7% homology with those of the dog or cat. All of 4 testes samples used in this study showed full spermatogenesis. Expressions of P450scc, 3βHSD and P450c17 mRNA were intense in the interstitial tissue, suggesting that pregnenolone, progesterone, and androstenedione were synthesized in the interstitial tissue. Signal of 17βHSD3 mRNA appeared at the margin of the seminiferous tubules. This is a remark that testosterone is synthesized inside the seminiferous tubules. P450arom mRNA expressed weakly inside almost entire parts of the seminiferous tubules. Estrogen would be synthesized not only in the somatic cells but also in the germ cells in bears, as reported in the mouse, rat, human, etc. These results were mostly in accordance with past reports on immunohistological localization of steroidogenic enzymes. Conclusion is that the interstitial tissue, probably Leydig cells, are the biosynthetic sites of steroid hormones such as pregnenolone, progesterone and androstenedione, and germ cells have capability for synthesizing estrogen in the Japanese black bear testes. Moreover, in bears, this report is the first detection of 17βHSD3, which is the isozyme specifically expressing in testes, and indicating that testosterone is synthesized inside the seminiferous tubules.