Compared with adult mammals, the brain of teleost fish is characterized by an extremely high capacity to aromatize androgens into estrogens, and this metabolic activity results from the expression of a specific brain aromatase (AroB) generated by the cyp19b gene. In this study, we first generated antibodies to zebrafish AroB and used them to map AroB-positive structures in the brain of adult zebrafish. We show that AroB is exclusively expressed in radial glial cells, mainly in the olfactory bulbs, telencephalon, preoptic area, and hypothalamus. Second, we investigated in vivo and in vitro the mechanisms involved in the estradiol (E2) regulation of the cyp19b gene. By means of whole-mount hybridization and immunohistochemistry on zebrafish embryos and larvae, we confirmed the E2-dependent upregulation of the cyp19b gene, and we show that E2 triggers AroB expression in radial glial cells mainly in the preoptic area and mediobasal hypothalamus of 48 hpf (hours post fertilization) and 108 hpf larvae. In addition, an in vitro analysis of 0.5 kb of the promoter region of the cyp19b gene demonstrated that this E2-dependent regulation involves a direct transcriptional action of estrogen receptors requiring estrogen-responsive elements. However, the data obtained on different cell lines demonstrate that a glial cell context is necessary for full E2 induction. The correlation between our in vivo and in vitro data suggests that the E2-dependent upregulation of AroB is favored by a glial cell context.