We have previously shown that neonatal exposure of rats to pharmacologic doses of diethylstilbestrol via daily injections resulted in a significant decrease in the estrogen-binding capacity of the uterine estrogen receptor (ER). In this study, we examined the effects of physiologic and pharmacologic doses of estradiol (E2) administered to adult ovariectomized rats via Silastic implants. Two days after implantation, uteri were removed, weighted, and homogenized, and ER levels were determined in the supernatant (hydroxylapatite assay) and low-speed pellet (nuclear exchange assay). Implants containing E2 concentrations of 0.005 or 0.05 mg/ml increased cytosolic but not total ER-binding capacity, whereas 0.5 or 5.0 mg of E2/ml implants decreased the binding capacity of cytosol ER to 40% and total ER to 50% of control values. The 0.005-mg/ml dose increased cytosol ER without increasing uterine weight; all higher doses significantly increased uterine weight. Determination of ER protein by an ER radioimmunoassay showed the same extent of reduction of ER concentration as the binding assays, demonstrating that the loss in E2 binding capacity is homologous down-regulation. The down-regulation of ER was maximal at 24 hr and was completely reversible after implant removal, although the time required to recover from down-regulation was dose dependent. Uterine weight also returned to control levels slowly after implant removal. Neither the sedimentation rate of the down-regulated ER nor the Kd of the cytosolic ER changed following long-term implantation; however, the Kd of the nuclear ER decreased significantly. This is the first demonstration of in vivo homologous down-regulation of uterine ER. ER down-regulation may play a role in several biologic processes.