The regulation of adenylate cyclase by muscarinic cholinergic receptors has been studied in rat heart membranes. In the presence of isoproterenol the K0.5 for Mg2+-mediated activation of adenylate cyclase was 0.1 mM; the addition of the muscarinic receptor agonist oxotremorine increased the K0.5 for Mg2+, and activation by Mg2+ no longer followed mass action kinetics for a single site interaction. The extent of oxotremorine-mediated attenuation of adenylate cyclase exhibited a Mg2+ concentration dependence: in short-time assays at 0.25 mM free Mg2+ the attenuation of enzyme activity was 55 percent, whereas at 20mM Mg2+, only 20 percent inhibition was observed. The increase in the apparent K0.5 for Mg2+ in the presence of oxotremorine was less in 20 min assays than in 4 min assays. The effects of oxotremorine on the rates of activation and deactivation of adenylate cyclase also were examined. Oxotremorine increased the rate of deactivation of adenylate cyclase by two-fold. At low free Mg2+ concentrations, oxotremorine also decreased the rate of activation of adenylate cyclase in the presence of either 0.1 uM GTP gamma S or 1 uM GTP. An oxotremorine-mediated decrease in the rate of activation of adenylate cyclase activity was not detectable in the presence of greater than 3 mM Mg2+.