To induce sex reversal of male to female, freshly-fertilized eggs of the S-rR strain medaka (Oryzias latipes) were immersed in saline containing estradiol-17β (E2) in different concentrations for various durations until hatching. Results of the present experiment showed that the immersion duration in 1 μg/ml E2 to induce 100% reversal of sex differentiation in the genotypic males was enough only for one day (24 hr) post-fertilization (dpf) and that treatment with E2 for 1 dpf resulted in a dose-dependent manner with the maximum sex reversal of 100% at 1 μg/ml. To ascertain early developmental periods efficacious for inducing sex reversal, additional brief immersion treatments of eggs with E2 were further performed individually for four different early developmental periods (Stages 4–9, 10–12, 13–15 and 16–18) within 1 dpf. As a result, induction of sex reversal was observed in all these short immersion periods without any restricted efficacy. Between both experimental and control groups treated with or without E2 for 1 dpf, differences in the number of germ cells in a gonad were compared in newly-hatched fry. It was found that gonads of the genotypic males (XY) treated with E2 revealed the female type which contained many germ cells with much dividing activity. These data suggest that a possible switch mechanism that exogenous E2 could trigger to change the genetic cascades involved in sex determination upon fertilization exists in early developmental stages.