Cytochrome P450c17 (CYP17, 17α-hydroxylase/17,20-lyase) is a critical enzyme in the production of androgens and estrogens in vertebrates. A 2102 bp full-length cDNA of P450c17-II (CYP17A2) has been isolated from the ovary of half-smooth tongue sole, Cynoglossus semilaevis which encodes 524 amino acids. The putative P450c17-II enzyme shares higher sequence identity with those of teleosts than with P450c17-I of vertebrate. The similarity between the two types of tongue sole P450c17 was 48%.Semi-quantitative RT-PCR analysis of spatial expression showed the enzyme was specifically expressed in the ovary and the head kidney. However, temporal expression shows that P450c17-II can be found in the brain. Furthermore, temporal expression pattern of P450c17-II in ovary and brain revealed developmental stage-dependency, and ovary P450c17-II expressed remarkably throughout the whole reproductive cycle. Otherwise, the expression pattern of P450c17-II in head kidney indicated negative ovary development-dependence. In addition, combined with our data on P450c17-I, T and E2 levels, the results further endorse the critical role of P450c17-II during shift in steroidogenesis, suggesting that P450c17-I and -II may act together to this physiological process. Based on the present study, we indicate an important role for P450c17-II during ovarian development.