The in vitro affinities to cytoplasmic estrogen receptors of the methylestrogens 2-methylestradiol-17 beta, 4-methylestradiol-17 beta and 4-hydroxy-2-methylestradiol-17 beta, which are useful probes to test the biological importance of 2- or 4-hydroxylation of estradiol-17 beta (catecholestrogen formation), have been determined in hypothalamic, pituitary and uterine tissue of the ovariectomized rat. Moreover, the in vivo capacity of these compounds to translocate estrogen receptors into the cell nucleus of pituitary and uterine tissue has been studied. Methylestrogens exhibited estrogen receptor affinities which were not significantly different from the binding affinity of estradiol-17 beta. When given at a high dose (100 micrograms/animal) their nuclear translocation capacity was equal (4-methylestradiol-17 beta) or even higher (2-methylestradiol-17 beta) than that of estradiol-17 beta. However, at a low dose (5 micrograms/animal) 4-methylestradiol was completely ineffective in both the pituitary gland and the uterus, and 2-methylestradiol-17 beta was less potent than estradiol-17 beta in the pituitary gland.