Uterine estrogen receptor (ER) and ER mRNA were measured in cycling and ovariectomized (OVX) estrogen-treated mice to probe the physiological regulation of the intracellular distribution and biosynthesis of ER. On proestrus, when plasma estradiol (E2) levels are highest, the cell nuclear ER concentration was 2.4-fold greater than on metestrus. This increase was primarily attributable to an increase in total cellular ER (cytosolic plus nuclear ER) and only secondarily to an activation of ER, as measured by its redistribution from the cytosolic (i.e. nuclear-extractable) to the nuclear (nonextractable) fraction. Total cellular ER concentration was 1.8-fold higher on proestrus than on metestrus, whereas the fraction of total ER in the nuclear compartment (i.e. the percentage activated) was only 1.3-fold higher. The concentration of cellular ER mRNA was 3-fold greater on proestrus than on the other days of the estrous cycle, suggesting that the increased concentration of ER on proestrus was a consequence of increased ER gene expression. In OVX mice, physiological and, to a lesser extent, supraphysiological levels of E2 increased cell nuclear ER. As in proestrous mice, the increased ER content contributed more than ER activation to the increased cell nuclear ER concentration. Physiological, but not supraphysiological, concentrations of E2 increased ER mRNA in OVX mice. Together, these results suggest that up-regulation by E2 of ER mRNA and ER accounts for most of the increased nuclear binding of ER on proestrus. E2-dependent activation and consequent DNA binding of ER presumably initiate this process, but quantitatively account for only a small fraction of the increased nuclear binding of ER.