Our previous studies showed that total nuclear estrogen receptor (Re) can be extracted and measured by exchange using 10 mM pyridoxal-5'-phosphate (PLP) at low temperature (0-4 degrees C). In order to further validate the PLP assay, we measured the Re concentration in uterine cytosol and nuclei by this method under physiological conditions, i.e. during the hamster estrous cycle and pseudopregnancy. In addition, we compared the Re results obtained by the PLP method with those obtained with two other assay procedures, i.e. the KCl and NaSCN methods. During the follicular phase of the estrous cycle, all three methods showed an elevation of nuclear Re in parallel with the increase in serum estradiol (E). However, the quantity of nuclear Re obtained with the PLP method was significantly greater than with the KCl method during the follicular phase. The surge of serum progesterone (P) during the ovulatory phase of the estrous cycle was followed by a dramatic fall in nuclear Re, and the greatest loss of nuclear Re during the ovulatory phase of the cycle was detected with the PLP and NaSCN methods. On a DNA basis, cytosol Re increased significantly between Day 3 and proestrus and subsequently fell during the ovulatory phase of the cycle. P treatment of proestrus hamsters resulted in a rapid (less than 4 h) loss of nuclear Re with little or no change in cytosol Re. Chronic P exposure during pseudopregnancy with serum E maintenance, resulted in a significant suppression of both cytosol and nuclear Re. Following P withdrawal, both cytosol and nuclear Re rose significantly (less than 4 h), indicating that this effect of P was readily reversible. The results demonstrate that the Re detected under physiological conditions by the PLP method responds to both E action and P action, and that the PLP assay provides a greater recovery of Re as compared to the KCl assay.