The presence of multiple monomeric forms of estrogen receptor (ER) has been described in different target tissues. Using [<sup>3</sup>H]tamoxifen aziridine (TA) to covalently label ER and SDS-PAGE to analyze labeled products, ER forms were investigated in ram pituitary and hypothalamus. A major labeled protein of M<sub>r</sub> 60,000-65,000 and a minor species of 50,000-55,000 were found in the pituitary cytosol covalently labeled with [<sup>3</sup>H]TA. In the hypothalamic cytosol, the major TA-labeled species was the M<sub>r</sub> 50,000 form while the 65,000 ER was difficult to detect. Comparison of ER forms after in vitro translocation of the ER complex in purified nuclei of ram pituitary or hypothalamus again showed major ER forms of M<sub>r</sub> 65,000 and 50,000 for the glandular and nervous tissue respectively, suggesting a biological significance for the M<sub>r</sub> 50,000 species. A similar heterogeneity was also observed in male rats used as controls. Moreover, covalent labeling of cytosol from the pars tuberalis/median eminence area showed the presence of ER in this part migrating with a pattern between those of the hypothalamus and the pituitary. The ER heterogeneity was thus demonstrated in the hypothalamo-pituitary axis. The source of this heterogeneity could be: (1) different ER mRNAs according to tissue type; (2) a specific posttranslational processing such as a specific proteolytic activity within the nervous tissue.