Administration of exogenous estradiol between stages 50 and 52 completely feminized the developing gonads of Xenopus laevis. However, when tadpoles were injected or cultured during the critical period with an inhibitor (CGS 16949A) of aromatase that prevents synthesis of estradiol from androgen, there were no detectable effects on the sexual differentiation of the gonads. Aromatase transcription in Xenopus gonads was then studied by the reverse-transcription polymerase chain reaction (PCR) method. In embryos at the beginning of the estradiol-sensitive period (stages 49 and 50), expression of the aromatase gene was not detected in the gonad. These results show that the period between stages 50 and 52 is the time when Xenopus is sensitive to sex reversal by estradiol and critical for sex determination, although estradiol synthesis may not be naturally involved in the gonad at this step.