- The binding of estrogen to endometrium and uterine arteries of the ewe was investigated by use of a [³H]estradiol exchange assay and by incubating whole tissue aliquants with [³ H]estradiol, a method which permits quantification of bound estrogen translocated from the cytoplasm to the nucleus. Five ewes were necropsied on each of days 0 (estrus), 3, 6 and 10 of the estrous cycle. Uterine horns and uterine arteries removed from ewes on days 3, 6 and 10 were identified as being either ipsilateral or contralateral to the ovary with the corpus luteum. Estradiol binding sites in endometrium were found to be saturable at ligand concentrations of 10 to 20 nM with optimal binding attainable following 60 min of incubation. The dissociation constant (Kd ) of the ligand-receptor complex was 8.30 nM and the maximum concentration of binding sites was determined to be 13,700 per cell. As determined by exchange assay, greater quantities of estradiol were bound to the endometrial cytoplasmic receptor (P < 0.01) on days 0 and 3 (6.84 and 9.56 fmoles/μg DNA, respectively) than on days 6 and 10 (1.59 and 2.80 fmoles/μg DNA, respectively). Nuclear-bound estradiol in endometrium averaged 1.09 ± 0.16 fmoles/μg DNA throughout the cycle. The concentration of specific nuclear-bound estradiol in endometrium from both uterine horns following translocation was greater (P < 0.01) on days 0 and 3 (3.03 and 3.75 fmoles/μg DNA, respectively) than on days 6 and 10 (1.69 and 1.64 fmoles/μg DNA, respectively). Nuclear-bound estradiol following translocation was greater in endometrium from the uterine horn ipsilateral to the ovary with the corpus luteum than in endometrium of the contralateral horn on day 10 only. Incubation of endometrium and uterine arterial segments from all ewes resulted in 77 ± 1% and 77 ± 5% of the estradiol bound in vitro being localized within the nuclear fractions, respectively. Estradiol in the nuclear fraction of both uterine arteries following translocation was greater (P < 0.01) on day 0 (0.35 ± 0.09 fmoles/pg DNA) than on days, 3, 6 and 10 (overall mean, 0.11 ± 0.02 fmoles/μg DNA). The concentration of nuclear-bound estradiol was greater in uterine arteries ipsilateral to the corpus luteum than in contralateral arteries on day 10 only. The effect of estrogen and progesterone on concentrations of estrogen receptor in endometrium of ovariectomized ewes was examined. Steroid treatment of ewes (four per group) for seven days was as follows: control (none), estradiol-17β(E²), progesterone (P⁴) and estradiol -178 and progesterone (E² + P4). Estradiol bound to cytoplasmic and nuclear receptors was greater (P < 0.05) in endometrium of E ² -treated ewes (10.40 ± 7.27 and 0.23 ± 0.06 fmoles /μg DNA, respectively) than in endometrium of control, P⁴- and E ² P ⁴ -treated ewes (1.62 ± 0.26 and 0.13 ± 0.02 fmoles/μg DNA, respectively). The concentration of [³H] estradiol specifically-bound in the cytoplasm and translocated to the nucleus during incubation was greater (P = 0.06) in endometrium of the E ² -treated animals (1.85 ± 0.43 fmoles/μg DNA) than in endometrium of the control, P⁴- and E² + P⁴- treated ewes (overall mean, 1.04 ± 0.19 fmoles/μg DNA). Similarly, the concentration of 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 the control, P⁴- and E² + P⁴-treated ewes (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 role of progesterone does not appear to involve the inhibition of estrogen-receptor complex translocation to the nucleus. Furthermore, it is proposed that uterine arteries may possess cytoplasmic receptors for estrogen similar to those present in endometrium.