Androgen and estrogen metabolism were examined in the period of steroid sensitivity during sex differentiation in mono-sex populations of Oreochromis niloticus. Fry (XX, XY, and YY genotypes) were maintained at 28° and were sampled at 8, 10, 11, and 13 days postfertilization. Sub-samples (n = 2-4) of pooled fry from each maternally distinct family were homogenized and incubated with either [3H]androstenedione or [3H]estradiol. Metabolites present in organic extracts were identified by thin-layer chromatography, microchemical reactions, and recrystallization to constant specific activity. Androstenedione was metabolized into at least seven readily identifiable compounds by all genotypes. In the XY genotype, 5β-androstane-3α,17β-diol synthesis decreased rapidly from 8 to 13 days postfertilization, with a concomitant increase in testosterone synthesis. Testosterone synthesis did not increase in the XX genotype. Testosterone synthesis in the YY genotype was intermediate to that of the XY and XX genotypes. Estrogens were not synthesized by any genotype. We hypothesize that 5β-reduction (or further hydroxylation) is a mechanism important in regulating testosterone production and subsequent sex differentiation. Results of incubations with estradiol show an age-dependent increase in metabolism which did not vary among genotypes. Metabolites synthesized included estrone and up to five unidentified compounds. © 2002 Elsevier Science (USA).