Cytochrome P450c17 (CYP17, 17alpha-hydroxylase/17, 20-lyase) has been viewed as a critical enzyme for the biosynthesis of gonadal and adrenal steroids in vertebrates. A full-length cDNA coding for P450c17 has been cloned from the zebrafish ovary. It encodes 519 amino acids and shares high sequence identity with that of fathead minnow (92%), rainbow trout (74%), chicken (64%), rat (47%), and human (48%). Northern blot analysis demonstrated that it is predominantly expressed in the ovary and testis; however, a weak hybridization signal was also detected in the whole kidney of the zebrafish, suggesting a role for P450c17 in the steroidogenesis of both gonads and renal/interrenal tissues. We further examined the expression of P450c17 in the developing ovarian follicles. Northern analysis and RT-PCR revealed abundant expression of the enzyme in the follicles of all major stages including pre-vitellogenic, vitellogenic and post-vitellogenic full-grown follicles; however, unlike P450c17 in the rainbow trout and eel, the expression of zebrafish P450c17 did not show significant temporal difference among different developmental stages, similar to the finding in the fathead minnow. The constitutive nature of P450c17 expression during zebrafish follicle development may partly explain the poor transcriptional response of the enzyme to the treatments with gonadotropin or activin, both of which enhance oocyte maturation in this species. It is also of interest to note that when assayed by RT-PCR, the enzyme P450c17 also seemed to be expressed in several other non-gonadal tissues including the brain, gill, liver and intestine, and its expression did not show significant sexual dimorphism in the brain of adult zebrafish. The physiological relevance of these findings remains to be further elucidated.