The administration of the steroid hormone, 17β-estradiol (E<sub>2</sub>) to ovariectomized rats increases the levels of muscarinic cholinergic receptors by 20–40% in the hypothalamic area. Using microdissection techniques, this increase has been shown to occur in hypothalamic subregions that possess high levels of intracellular receptors for E<sub>2</sub>. Among these regions is the ventromedial nucleus (VMN), which may be the principal target site in the rat brain for the activation of feminine sexual behavior by E<sub>2</sub>. In this study, we have further characterized the increase in muscarinic binding within the VMN after E<sub>2</sub> and show that it satisfies many of the criteria for an important regulatory mechanism by which E<sub>2</sub> could activate sexual behavior: (1) Using quantitative receptor autoradiography, it was determined that the increase in [<sup>3</sup>H]-QNB binding in the VMN after E<sub>2</sub> results from an increase in the number of receptors rather than a change in the affinity of the binding. (2) The increase in VMN muscarinic receptors as determined by the binding of [<sup>3</sup>H]-QNB to microdissected homogenates occurred by 18–24 h after exposure to E<sub>2</sub>, the earliest time at which sexual behavior is facilitated. (3) Increased muscarinic receptors did not occur in the VMN of male rats, which show little activation of feminine sexual behavior after E<sub>2</sub> exposure. (4) Muscarinic receptors in the VMN of female rats are induced at 24 h by a 6 h exposure to E<sub>2</sub>, the minimum length of time that is sufficient for E<sub>2</sub> to activate sexual behavior. A 4 h exposure to E<sub>2</sub> is insufficient to activate both sexual behavior and induce muscarinic receptors. (5) Administration of the protein synthesis inhibitor, anisomycin, during the 6 h E<sub>2</sub> exposure will block both the increase in muscarinic receptor levels and the activation of feminine sexual behavior. These results are consistent with the notion that the muscarinic cholinergic receptor may be one of the proteins induced by E<sub>2</sub> in the VMN to activate sexual behavior.