Androgens and especially 11-oxygenated androgens are known to be potent masculinizing steroids in fish. As a first step to study their physiological implication in gonadal sex differentiation in fish, we cloned a testicular cytochrome P450(11beta) (11beta-hydroxylase) cDNA in the rainbow trout, Oncorhynchus mykiss. We isolated a 1882 bp P450(11beta) cDNA (rt11betaH2, AF217273) which contains an open reading frame encoding a 552 putative amino acids protein. This sequence was highly homologous (98% in nucleotides and 96.5% in amino acids) to another rainbow trout P450(11beta) sequence (AF179894) and also to a Japanese eel P450(11beta) (68% in amino acids). Northern blot analysis detected a single transcript of 2 kb which was highly expressed in the testis (stage II) and to a lesser degree in the anterior kidney (containing the interrenal tissue). No signal was detected in the posterior kidney, brain, liver, skin, intestine and heart. In the testis this transcript was highly expressed at the beginning of spermatogenesis (stages I and II), followed by a decrease during late spermatogenesis (stages III to V). By semi-quantitative reverse transcription polymerase chain reaction, P450(11beta) expression during gonadal differentiation was estimated to be at least 100 times higher in male than in female gonads. This difference was first detected at 55 days post-fertilization (dpf), i.e. 3 weeks before the first sign of histological sex differentiation, and was sustained long after differentiation (127 dpf). Specific P450(11beta) gene expression was also demonstrated before testis differentiation (around 50 dpf) using virtual Northern blot, with no expression detected in female differentiating gonads. From these results, and also based on the already known actions of 11-oxygenated androgens in testicular differentiation in fish, it is now suggested that P450(11beta) gene expression is a key factor for the testicular differentiation in rainbow trout.