We have developed and validated assay methods which are appropriate for studying the subcellular distribution of estrogen receptor (Re) and progesterone receptor (Rp) in hamster uterus during the estrous cycle and pregnancy. Cytosol receptors were monitored by conventional procedures, and nuclear receptors were extracted from nuclei at 2 C using 0.5 M KCl in buffer containing glycerol and measured by ligand exchange during incubation with [3H] progesterone (P) at 2 C for 24 h or [3H] estradiol-17 beta (E2) at 30 C for 1 h. The nuclear exchange assay described herein measures total receptor and also permits estimation of unlabeled steroid in the nuclear KCl extract. Nuclear Rp translocation was shown to be target-tissue specific, hormone specific, and dependent on P dosage under in vivo conditions. The duration of nuclear Rp retention was about 6 h following exogenous P treatment. During the estrous cycle, Re and Rp levels in uterine cytosol and nuclei varied in response to the changing pattern of E2 and P secretion. Our results document a positive relationship between serum E2, nuclear Re and cytosol Rp levels during the follicular phase of the cycle. Nuclear Rp remained low on cycle days 1--3, and increased abruptly on day 4 coincident with preovulatory P secretion and cytosol Rp depletion. Nuclear Rp translocation during the preovulatory period on cycle day 4 was associated with a marked decline in nuclear Re, and the latter was demonstrated to be a rapid (2--4 h) response to P action rather than serum E2 withdrawal. The negative effect of P on nuclear Re levels may represent a fundamental mechanism for P antagonism of E action. Cytosol and nuclear Rp levels in deciduomal and myometrial tissue were responsive to experimental alteration of serum P, indicating that nuclear Rp is maintained by a continuous process of P-induced Rp translocation. Of interest was the finding in pregnant animals that Re levels declined dramatically in deciduoma as compared to myometrium. The loss of cytosol and nuclear Re from deciduomal cells cannot be attributed to changes in serum E2 or P. Thus, the intracellular regulation of Re numbers may change during differentiation of certain target cells such as those found in the deciduoma.