Pregnant ICR/JCL mice were treated with 6 mg of cyproterone acetate (CA) from days 14 to 20 of pregnancy to feminize male offspring. Feminized males delivered on day 20 of pregnancy by cesarean section were castrated the same day, injected with estradiol-17beta(E2) or sesame oil from the day of delivery (=day 1) to day 10 and sacrificed on day 60. In oil-injected feminized males, the vaginal epithelium was atrophic and did not show cornification. In feminized males given 20 microgram E2 neonatally, the vaginal epithelium exhibited well-differentiated stratified squamous organization, but was not cornified in seven out of the nine mice of this group. In the mice treated with 50 microgram E2, persistent cornification was recognized most frequently in the posterior two-thirds of the vaginal epithelium which is considered to originate from the urogenital sinus. However, the incidence of cornification in the anterior one-third which may contain the epithelial cells of müllerian duct was low. These results provide supporting evidence for the possible participation of epithelial cells which come from the urogenital sinus in the development of estrogen-independent persistent vaginal cornification in neonatally estrogenized mice.