There are two patterns of gonad differentiation in amphibians, but the role of sex steroids in gonad differentiation is not clear. I studied the role of estrogen in gonadal sex differentiation in the frog Euphlyctis cyanophlyctis with an undifferentiated type of gonad differentiation (testis differentiates through an ovarian phase) using an aromatase inhibitor, formestane (FR). I treated tadpoles with four concentrations of FR (1, 10, 50, and 100 μg/L) during Gosner stages 25–42. Treatment of higher concentrations (50 and 100 μg/L) of FR produced a male-biased sex ratio and inhibited ovary development but not ovarian cavity and meiocytes formation. These results suggest that estrogen may not be involved in early ovarian differentiation and the ovarian phase is important for testis differentiation in E. cyanophlyctis.