The effect of Ca2+ on the biochemical characteristics of muscarinic receptors in the adenohypophysis of male and female rats at the various stages of the estrous cycle was investigated in binding experiments using the specific muscarinic antagonist N-methyl-4-piperidyl benzylate ([3H]-4NMPB) and the muscarinic agonist oxotremorine. By using Ca2+ chelators such as EGTA, and Ca2+ channel blockers such as D-600, we showed that Ca2+ profoundly alters the binding characteristics of both antagonists and agonists to the muscarinic receptors. In female rats the effect of Ca2+ on antagonist binding is mainly on the maximal binding capacity of the receptors, while changes in the dissociation constants are much more moderate. The effect is expressed in the ability of Ca2+ to expose or to eliminate binding sites as a function of the estrous cycle. In agonist binding, the presence of Ca2+ has a pronounced effect on the proportion of high-affinity binding sites, which parallels the changes induced in antagonist binding throughout the estrous cycle. Interestingly, the natural progression of the cycle from diestrus 2 to the estrous stage undergoes a change identical to that occurring in vitro upon Ca2+ removal. D-600 can completely block the effect of Ca2+ on the binding of both [3H]-4NMPB and oxotremorine. The concentration of D-600 required in order to induce such blocking is also dependent on the estrous cycle. It appears that the progression of the estrous cycle is accompanied by changes in the muscarinic receptors which may in turn be coupled to Ca2+ channels.