Meiotic maturation in fish is accomplished by maturation-inducing hormones. 17α,20β-Dihydroxy-4-pregnen-3-one (17α,20β-DP) was identified as the maturation-inducing hormone of several teleosts, including Nile tilapia. A cDNA encoding 20β-hydroxysteroid dehydrogenase (20β-HSD), the enzyme that converts 17α-hydroxyprogesterone to 17α,20β-DP, was cloned from the ovarian follicle of Nile tilapia. Genomic Southern analysis indicated that 20β-HSD probably exists as a single copy in the genome. The Escherichia coli-expressed cDNA product oxidized both carbonyl and steroid compounds, including progestogens, in the presence of NADPH. Carbonyl reductase-like 20β-HSD is broadly expressed in various tissues of tilapia, including ovary, testis, and gill. Northern blot and reverse transcription polymerase chain reaction analyses during the 14-day spawning cycle revealed that the expression of 20β-HSD in ovarian follicles is low from Day 0 to Day 8 after spawning and is not detectable on Day 11. Distinct expression was evident at Day 14, the day of spawning. In males, 20β-HSD expression was observed continually in mature testes but not in immature testes of 30-day-old fish. In vitro incubation of postvitellogenic immature follicles (corresponding to Day 11 after spawning) with hCG induced the expression of 20β-HSD mRNA transcripts within 1–2 h, followed by the final meiotic maturation of oocytes. In tissues such as gill, muscle, brain, and pituitary, however, hCG treatment did not induce any changes in the levels of mRNA transcripts. Actinomycin D blockade of hCG-induced 20β-HSD expression and final oocyte maturation demonstrated the involvement of transcriptional factors. The carbonyl reductase-like 20β-HSD plays an important role in the meiotic maturation of tilapia gametes.