The highly specific tritiated muscarinic antagonist N-methyl-4-piperidyl benzilate [(3H)-4NMPB] was used in direct binding and in competition experiments with the muscarinic agonist oxotremorine to investigate the influence of in vivo endocrine manipulations involving estrogen on muscarinic receptors in the adenohypophysis and hypothalamus. In the adenohypophysis the characteristics of antagonist binding in ovariectomized female rats, like those in androgenized females, resembled those in normal male rats; this trend was reversed in ovariectomized females after implantation of 17 beta-estradiol capsules, with the characteristics of antagonist binding now resembling those in normal female rats at estrus. Agonist binding characteristics also showed some distinct differences between treated and normal female rats: the proportion of high affinity binding sites decreased both in ovariectomized and androgenized rats, while the affinity of these sites became greater as compared to control animals. The results clearly show that changes in the levels of sex steroids, as a result of the above-mentioned endocrine manipulations, influence the behavior of the muscarinic receptors in both areas, and suggest muscarinic participation in the regulation of gonadotropin release in the hypothalamus-pituitary-ovary axis.