On Day 5 of incubation fertilized eggs of single-comb White Leghorn hens were injected with an aromatase inhibitor (AI) and the sex reversal effect and levels of mRNA of P45017α-hydroxylase (P45017α) and P450aromatase (P450arom) were evaluated by observation of gonadal phenotype and by Northern and slot blot analysis. Individual genetic sex was evaluated by Southern blot analysis of red blood cells using a female sex chromosomal W-specific DNA probe. Saline injection had no sex reversal effect, whereas AI injection resulted in 50% sex reversal from genetic female but no effect on male. Levels of P45017α mRNA were high in both ovary and testis in the control group but these levels were lowered significantly in ovary, testis, and sex-reversed gonad (testis) in the AI-treated group. On the other hand, levels of P450arom mRNA in the ovary were higher than those in the testis of the control group. AI treatment significantly suppressed ovarian levels of P450arom mRNA. Although estradiol alone failed to prevent the phenotypic male to female change, coadministration of estrogen suppressed the sex-reversal effect of AI and restored mRNA levels of P450arom in the ovary to control levels. These results suggest that expression of P450arom mRNA and estrogen plays an important role in sex differentiation of the female gonad of the Chicken.