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17 alpha, 20 beta-Dihydroxy-4-pregnen-3-one (DHP) is a maturation-inducing steroid in many teleost fish. Carbonyl reductase-like 20 beta-hydroxysteroid dehydrogenase (CR/20 beta-HSD) is a candidate enzyme responsible for DHP production during oocyte maturation in various fish, including Nile tilapia. However, a novel type of 17 beta-hydroxysteroid dehydrogenase, type 12-like (17 beta-HSD12L), is responsible for DHP production during oocyte maturation in masu salmon. 17 beta-HSD12 (presumably orthologous to salmon 17 beta-HSD12L) has been detected in Nile tilapia; however, its enzymatic activity and specific ability to convert the DHP substrate 17 alpha-hydroxyprogesterone (17OHP) have not been examined. This study aimed to determine whether CR/20 beta-HSD or 17 beta-HSD12 is responsible for DHP production during oocyte maturation in the Nile tilapia. Mammalian expression vectors containing tilapia hsd17b12 or CR/20bhsd were transfected into HEK293T cells, followed by incubation with 170HP. HEK293T cells transfected with hsd17b12 exhibited a strong ability to convert exogenous 17OHP to DHP (73.8% yield). Cells transfected with CR/20bhsd or the control vector converted only 7.4% and 7.5% of 17OHP to DHP, respectively. In addition, based on LC-MS/MS analyses, 17 beta-HSD12 did not convert any substrates other than 17OHP, including DHP, adrenosterone, androstenedione, estrone, testosterone, 11-ketotestosterone, and estradiol-17 beta. CR/20 beta-HSD showed strong 17 beta-HSD oxidoreductase activity especially with adrenosterone and androstenedione. Tissue-specific hsd17b12 expression analyzed by RT-PCR showed that hsd17b12 mRNA was strongest amplification in full-grown follicles. Finally, full-grown ovarian follicles were incubated with salmon pituitary extract (SPE, 100 mu g/mL) or human chorionic gonadotropin (HCG, 100 IU/mL) to induce 2013-HSD activity in vitro, and enzyme activity was assessed by co-incubation with 100 ng/mL 17OHP for 2, 4, 8, and 16 h. Conversion of