Estrogen formation catalyzed by neural aromatase is crucial for the sexual differentiation of the brain. Ontogenic expression of aromatase mRNA and aromatase activity were studied in male and female rat midbrains. Aromatase mRNA was transiently expressed in both sexes showing maximum levels on postnatal day (P)2 and being absent on P20 and in adults. Developmental expression of aromatase mRNA preceded that of aromatase activity. These data demonstrate that the capacity for estrogen formation is present during a distinct phase of midbrain development. Our findings suggest an active role for estrogens in the differentiation of midbrain neurons.