This study determined whether the biochemical characteristics of the estradiol (E2) nuclear receptor located in three different brain areas and the pituitary gland (PIT) change in aging rats undergoing declining reproductive function. To measure maximal E2 nuclear receptor concentrations, groups of young (3-4 months old), middle-aged (8-11 months old), and old (16-18 months old) cycling rats were injected iv with E2 to translocate maximally cytosolic receptors into the nucleus. One hour later they were killed and nuclear extracts were prepared. Maximal E2 nuclear receptor concentrations and dissociation constants were assessed in the preoptic area (POA), medial basal hypothalamus (MBH), amygdala (AMYG), and PIT using an in vitro exchange assay and covariance analyses of double reciprocal plots (Lineweaver-Burk). Middle-aged cycling rats exhibited decreased E2 nuclear receptor concentrations in the POA, but no change in the MBH, AMYG, or PIT. Old rats exhibited decreased E2 nuclear receptor concentrations in the POA, MBH, and PIT and an unexpected increase in the AMYG. There was no change in the affinity of the receptor with age, although an apparent decrease in the pituitary E2 nuclear receptor of middle-aged rats was observed. This difference was not detected when saturation analyses were performed using varying dilutions of the pituitary nuclear extract. These results demonstrate that changes occur in maximal numbers of E2 nuclear receptors in the POA of middle-aged rats. As animals age further, the changes encompass a larger brain area and include the PIT. The data suggest that changes in E2 nuclear receptor concentrations may contribute to the age-related decline in reproductive function.