The in vitro effects of estrogens, progestins, and their related analogs on muscarinic receptor binding sites were studied in the hypothalamic membranes prepared from ovariectomized rats. The binding assays were performed under a nonequilibrium condition. Progestins and their metabolites were active in inhibiting the binding of [3H](-)QNB to muscarinic receptors, whereas estrogenic compounds were devoid of this effect. Progesterone was also found to be active in inhibiting the binding of [3H](-)QNB to pituitary membranes. The IC50 values of progesterone and its metabolite, 17α-hydroxyprogesterone, were 34 and 24 µM, respectively. The inhibitory effect of progesterone was rapid, reversible, and not dependent on divalent metal ions (Ca2+, Cu2+, Fe2+, Mg2+, Mn2+, and Zn2+). Analyses of the binding data with Scatchard and Lineweaver-Burke plots revealed that progesterone significantly increased the apparent Kd of muscarinic receptor binding sites from 0.54 (SE= ± 0.08) nM to 2.44 (SE= ± 0.49) nM in hypothalamic membranes and from 0.21 (SE= ± 0.03) nM to 0.34 (SE= ± 0.03) nM in pituitary membranes without a significant effect on the receptor density in both membrane preparations. Progesterone decreased the rate of association of [3H](-)QNB with muscarinic receptors without a significant effect on its rate of dissociation from the [3H](-)QNB-receptor complex. These results indicate that progesterone, but not estrogenic compounds, was capable of interacting with hypothalamic and pituitary muscarinic receptors in