The mechanism by which progesterone antagonizes estrogenic stimulation of uterine growth was examined in the immature rat. Rats received daily injections of 2.5 mug estradiol (E) for 2 days and on day 3 either 2.5 mug E or 2.5 mug E plus 2.5 mg of progesterone (P). The quantity of nuclear and cytoplasmic estrogen receptor was determined by [3H]estradiol exchange at various intervals after injection of E or E + P. In both groups, nuclear receptor estrogen complex (RnE) increased dramtically one hour after injection and showed a gradual decline from 4 to 24 h after injection. The quantity of cytoplasmic receptor, Rc, decreased to low levels by one hour and began a gradual increase from 4 to 8 h in both groups. However, between 8 and 24 h after injection, the level of Rc continued to increase in the E treatment group (2.39 +/- 0.21 pmol/uterus at 24 h) but remained at the 8 h level in the E + P group (1.09 +/- 0.04 pmol/uterus at 24 h). This observation suggests that two seperate processes are involved in the replenishment of Rc and that progesterone inhibits the second phase of replenishment. The binding affinity and specificity of Rc for estrogens following E + P pretreatment were identical to those of the E pretreatment group. Therefore, P does not alter the binding properties but rather the intrauterine level of Rc. Treatment with E on day 4, when Rc levels differ between E and E + P groups, stimulated uterine weight and protein content on day 5 in the E pretreatment group. However, minimal stimulation was observed in the E + P pretreatment group. The quantity of RnE and the time of nuclear retention of RnE following E injection on day 4 was greater in the E group than in the E + P group. The effect of progesterone on Rc replenishment was dose-dependent (range, 0.1-2.5 mg; 1/2 maximal, 0.5 mg). Injection of testosterone propionate (1.0 mg), a weak estrogen antagonist, with E on day 3 resulted in slightly reduced levels of Rc on day 4. This reduction also correlated with a reduced sensitivity to treatment with E on day 4. These data, together with previous studies from our laboratory, suggest that progesterone and other estrogen antagonists such as nafoxidine and testosterone propionate inhibit estrogen action by interfering with the replenishment of Rc, thereby reducing the number of receptor estrogen complexes that are translocated and retained by uterine nuclei.