Sheep exposed to testosterone during a critical period from gestational day (GD) 30 to GD 90 develop masculine genitals and an enlarged male-typical ovine sexually dimorphic nucleus of the preoptic area (oSDN). The present study tested the hypothesis that separate critical periods exist for masculinization of these two anatomical end points. Pregnant ewes were treated with testosterone propionate (TP) either from GD 30 to GD 60 (early TP) or GD 60 to GD 90 (late TP). Control (C) pregnant ewes were treated with corn oil. Fetuses were delivered at GD 135 and the volume of the oSDN was measured. Early TP females possessed a penis and a scrotum devoid of testes, whereas late TP and C females had normal female genitals. Neither period of TP exposure grossly affected the genitals of male fetuses. Despite masculinized genitals, the mean volume of the oSDN in early TP females (0.32 ± 0.06 mm³) was not different from C females (0.24 ± 0.02 mm³) but was significantly enlarged in late TP females (0.49 ± 0.04 mm³; P < 0.05 vs. C) when the genitals appeared normal. In contrast, the volume of the oSDN in late TP males (0.51 ± 0.02 mm³) was not different from C males (0.51 ± 0.04 mm³) but was significantly smaller in the early TP males (0.35 ± 0.04 mm³; P < 0.05 vs. C). These results demonstrate that the prenatal critical period for androgen-dependent differentiation of the oSDN occurs later than, and can be separated temporally from, the period for development of masculine genitals.