We performed assays for aromatase activity in serial 200 μm sections of the three orthogonal planes of the brain and produced a three dimensional reconstruction of aromatase levels in Medaka (Oryzias latipes) brain. The regulation of this brain aromatase activity by sex steroids was then examined by in vivo exposure bioassays. We demonstrate that the activity of brain aromatase is localized to an area that contains the hypothalamus, a region of the brain involved in the regulation of the reproductive function. The exposure of Medaka to estradiol lead to increased brain aromatase levels; whereas, exposure to methyl-testosterone lead to decreased brain aromatase levels. The regulation of brain aromatase levels by both product and substrate may provide a sensitive trigger for environmental cues to alter sex behavior.