Steroid binding in both the vaginal epithelium and the vaginal fibromuscular wall (FMW) was compared in control and neonatally estrogen-treated mice. Neonatal treatment with a low dose of the estrogen diethylstilbestrol (DES) had no significant effect on adult estrogen binding within the assayed vaginal compartments; however, this treatment caused a 2-fold increase in the level of cytosolic progestin binding in the vaginal FMW over that in vehicle-treated mice. This low neonatal dose did not affect the level of progestin binding in the vaginal epithelium. In contrast, neonatal treatment with a larger dose of DES caused marked increases in cytosolic progestin binding, decreases in cytosolic estrogen binding, and increases in nuclear estrogen binding within the FMW. Furthermore, as a result of the changes in specific binding induced by the neonatal DES treatment, the degree of the estrogen binding within in each tissue shifted from a predominantly cytosolic site to a nuclear one.