20 beta-Hydroxysteroid dehydrogenase (20 beta-HSD) is a crucial enzyme that converts 17 alpha-hydroxyprogesterone to 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP), which triggers oocyte maturation in most teleost fish. A full-length cDNA for a carbonyl reductase-like 20 beta-HSD (CR/20 beta-HSD) has been cloned from the zebrafish ovary. Although the zebrafish CR/20 beta-HSD is expressed in all of the tissues tested, it is predominantly expressed in the ovary, testis, kidney, and gill. In the ovary, the enzyme was shown to be expressed in the follicle cells and its expression appeared to be constitutive. No significant difference was noticed in the level of CR/20 beta-HSD expression among follicles of different stages. Furthermore, analysis of the ovarian samples taken at different times before spawning showed no significant change of the enzyme expression. In agreement with these results, treatment of the cultured zebrafish ovarian follicle cells with gonadotropin and activin had little effect on the expression of the enzyme. Taken together, these results point to the possibility that the gonadotropin-induced DHP production and final oocyte maturation in the zebrafish may not involve significant change of CR/20 beta-HSD expression as evidenced in the salmonids, or that there might be other isoforms of 20 beta-HSD whose expression is tightly controlled by endocrine and paracrine factors.