The relationship between the steroid hormone 17β-estradiol and the muscarinic cholinergic receptors present in the preoptic area (POA), median hypothalamus and posterior hypothalamus of female rats was examined in vitro at various stages of the estrous cycle. Muscarinic receptors varied in a cyclic manner, specifically in the POA, as shown by an increase in the proportion of high-affinity agonist binding sites (RH) to 60% during the proestrus, as compared to (RH) proportion observed during diestrus-2 and during the afternoon of proestrus (35%). Exposure of POA homogenates to 17β-estradiol resulted in conversion of (RH) to low-affinity agonist binding sites (RL) This effect of the hormone was also restricted to the POA taken from rats during the morning of proestrus. It was blocked by the antiestrogenic drug, clomi-phene, and could be prevented by preoccupation of the muscarinic receptors by their own ligands prior to the addition of hormone. It follows that significant changes in POA muscarinic receptors in situ exactly coincided with the known critical time period characterized by high estrogen levels and high levels of estrogen receptors in the POA. These changes in muscarinic receptors might thus conceivably reflect variations in cholinergic activity in the POA during the estrous cycl