A series of experiments was carried out in which genetically female Nile tilapia (Oreochromis niloticus) fry were treated with Fadrozole, a nonsteroidal aromatase inhibitor (AI), in the diet during the period of sexual differentiation. Batches of tilapia fry treated with AI during the first 30 days following yolk-sac resorption (7–37 days post hatch, dph) showed a dose-dependent increase in the percentage of males from 0 to 200 mg · kg–1. The percentage of males remained approximately constant (92.5–96.0%) from 200 to 500 mg · kg–1. Any continuous 2- or 3-week treatment with 500 mg · kg–1 AI in this 4-week period successfully masculinized the majority of the treated fish (>80%). Treatments of 1 week duration revealed that the most sensitive time to AI lies in the first week (between 7 and 14 dph). Progeny testing of males from AI-treated groups gave results indicating that these were XX males, as expected. These experiments strongly implicate aromatase activity as a key factor in sexual differentiation in the Nile tilapia. J. Exp. Zool. 287:46–53, 2000. © 2000 Wiley-Liss, Inc.