Progesterone normally antagonizes vaginal cornification resulting from estrogen treatment. We, therefore, examined the effects of in vivo progesterone treatment on retention of [3 H] estradiol by vaginal tissue from normal and neonatally androgen or estrogen treated rats. Progesterone treatment in vivo resulted in a significant increase in [3 H] estradiol retention by vagina at 48, 72, 96 and 120 h in the continued presence of the progesterone source. Following 24 h of progesterone treatment, a significantly increased retention of [3 H] estradiol could be measured at 36, 48, 60 and 72 h after removal of the progesterone source. Rats receiving estradiol benzoate or testosterone propionate neonatally differed significantly from controls by retaining less [3 H] estradiol and the neonatally hormone treated animals failed to show increased [3 H] estradiol retention following in vivo progesterone treatment. [3 H] Estradiol retention was also measured in vitro following homogenization of vaginal tissue and incubation at 30 or 37° C for 1 h in 0.01 µM [3 H] estradiol. Vaginal tissue from control animals treated for 72 h with progesterone showed a significant enhancement of estrogen retention by the nuclear fraction. Incubation of the cytosol fraction from the progesterone treated animals at 0-4° C indicated a significant decrease in estrogen retention. Thus the increased nuclear binding of estrogen observed in the progesterone treated animals cannot be explained by a progesterone induced increase in cytosol estrogen receptor. The mechanism of the progesterone induced increase in nuclear retention of estrogen is presently unknown. However, this mechanism appears not to be operative in animals which received neonatal treatments with estradiol benzoate or testosterone propionate.