A study was conducted to examine the influence of estradiol and progesterone on total cytoplasmic and nuclear estrogen receptor concentrations in endometrium of ovariectomized ewes. Steroid treatment of ewes (four per group) for 7 days was as follows: Control (none), estradiol-17β (E2, 54 mg implant), progesterone (P4, 15 mg twice daily) and estradiol and progesterone (E2 + P4). Concentrations of estrogen receptor were determined by exchange assay and after incubation of tissue with [3 H] estradiol for 1 h. As determined by exchange assay [3 H] estradiol bound to total cytoplasmic and nuclear receptors was greater (P2-treated ewes (10.40 ± 7.27 and 0.23 ± 0.06 fmoles/µg DNA, respectively) than in endometrium of control, P4- and E2 + P4-treated animals (1.62 ± 0.26 and 0.13 ± 0.02 fmoles/µg DNA, respectively). An average of 81 ± 1 percent of the [3 H] estradiol bound to available cytoplasmic receptors was translocated to the nucleus during incubation of endometrium of ewes from all treatment groups. The concentration of [3 H] estradiol specifically-bound in the cytoplasm and translocated to the nucleus during incubation tended to be greater (P=0.06) in endometrium of the E2-treated animals (1.85 ± 0.43 fmoles/µg DNA) than in endometrium of ewes in the control, P4-treated and E2 + P4-treated groups (overall mean, 1.04 ± 0.19 fmoles/µg DNA). Similarly, the concentration of labeled estradiol bound in the cytoplasm after in vitro translocation was greater in endometrium of E2-treated ewes (0.49 ± 0.10 fmoles/µg DNA) than in endometrium of ewes in the control, P4- and E2 + P4-treated groups (overall mean, 0.26 ± 0.06 fmoles/µg DNA). These data suggest that progesterone antagonizes estrogen binding in the uterus by decreasing the concentration of cytoplasmic estrogen receptor. The suppressive action of progesterone does not appear to involve inhibition of estrogen-receptor complex translocation to the nucleus.