Two cDNA clones of cytochrome P-450 aromatase (P-450arom) were isolated from a medaka (Oryzias latipes, a daily spawner) ovarian follicle cDNA library using a medaka P-450arom genomic DNA fragment as a probe. The first, 1,809-bp insert (S81f) contains an 1,554-bp open reading frame encoding a 518-amino-acid polypeptide. The second, 1,852-bp (S52f) insert possesses an open reading frame identical to that of the S81f insert, except for the absence of the heme-binding region as the result of an additional A residue at the position of nucleotide 1,827. Expression of the S81f cDNA, but not of the S52f cDNA, in nonsteroidogenic COS-1 cells leads to production of a steroidogenic enzyme which is capable of converting exogenous testosterone to estrogen. The P-450arom genomic DNA fragment hybridizes to a single mRNA in medaka ovarian follicle RNA. Changes in level of P-450arom transcripts and P-450arom enzyme activity were determined in medaka ovarian follicles collected at 16 stages of development. A close correlation was found between these two profiles, both being high in midvitellogenic follicles and low in postvitellogenic follicles collected during oocyte maturation. These findings, together with those of our previous study showing that actinomycin D prevented gonadotropin-induced aromatase activation by medaka ovarian follicles, suggest that aromatase activity is regulated at the transcriptional level in medaka vitellogenic follicles.