A cDNA clone encoding 3 beta-hydroxysteroid dehydrogenase delta 5-4-isomerase (3 beta-HSD) was isolated from a cDNA library of rainbow trout ovarian thecal cells. Southern hybridization analysis of trout genomic DNA with the cDNA suggested the presence of a single gene encoding 3 beta-HSD in the rainbow trout showing a total genomic size of less than 4 kilobases (kb). The cDNA hybridized to a species of mRNA isolated from rainbow trout ovaries; the 1.4 kb transcripts were most abundant in trout ovaries during the later stages of oogenesis. The trout 3 beta-HSD expressed in non-steroidogenic monkey kidney tumor (COS-1) cells showed a unique enzymatic 3 beta-HSD activity. Dehydroepiandrosterone was a more favoral substrate of the trout 3 beta-HSD than 1- alpha-hydroxypregnenolone. Interestingly, the trout 3 beta-HSD expressed in COS-1 cells exhibited minimal ability to convert pregnenolone to progesterone.