We isolated a novel type of aromatase cDNA from a Nile tilapia (Oreochromis niloticus) ovary cDNA library. Because this aromatase is phylogenetically related to brain aromatase (CYP19b) of goldfish, zebrafish and sea bass, we named it tilapia CYP19b (tCYP19b). tCYP19b encodes a protein that is predicted to consist of 495 residues and have 63.8% homology with the aromatase (tCYP19a) we previously isolated from the same source. In vitro transient transfection of cultured COS7 cells demonstrated that tCYP19b codes a functional protein to catalyze estrogen production from an androgen substrate. RT-PCR and Northern hybridization analysis showed that tCYP19b was expressed at a high level in the brain and at a low level in a wide variety of other tissues, whereas tCYP19a was mainly present in the ovary and its level significantly increased during the vitellogenic stage. RT-PCR also detected tCYP19b expression in brain and gonad tissues of both female and male tilapia during sex differentiation, but tCYP19a was only found in the ovary of the fry at that period. These results suggest that tCYP19a plays a key role in sex differentiation and ovarian development. We also isolated genes of two tilapia aromatases. Based on the location of the transcription initiation site, we predicted that there is one promoter for tCYP19a and three promoters for tCYP19b. Although the two aromatase isoforms have similar gene structures in the coding region, we found that the binding regions of SF-1/Ad4 BP region, WT1-KTS and SRY, which are sex-determining factors in mammals, are present in the 5' flank region of tCYP19a but not tCYP19b. A similar situation is present in promoters of zebrafish and goldfish aromatase isoforms. This data indicates that CYP19a plays a decisive role in sex differentiation of those species. The unique presence of the ERE motif in the tCYP19b promoter and the high expression of tCYP19b in the brain support that CYP19b is mainly involved in estrogen-mediated neural estrogen synthesis.