Rat myocardial adenylate cyclase activity was characterized with respect to its regulation by Mg and Mn. Myocardial adenylate cyclase is activated by both free divalent cations at concentrations greater than that required to form the active cation-ATP substrate. Six separate lines of evidence indicate that Mg and Mn are distinguishable from each other as regulators of adenylate cyclase: 1) After solubilization, adenylate cyclase is preferentially stimulated by Mn. 2) High concentrations of Mn (greater than 1 mM)--but not Mg--have an additional effect on particulate adenylate cyclase to attenuate basal adenylate cyclase activity and to render the membranes refractory to stimulation by isoproterenol and guanyl-5'-yl-imidodiphosphate [Gpp(NH)p]. This inhibitory property of Mn is lost after solubilization. 3) Isoproterenol enhances the affinity of adenylate cyclase for Mg but not for Mn. 4) Stimulation of adenylate cyclase by Gpp(NH)p is consistent with a simple single-site interaction of Gpp(NH)p with the adenylate cyclase complex in the presence of Mg. In contrast, Mn exposes a broad, 3-log-order, stimulatory response to Gpp(NH)p, 0.1 to 100 microM. After solubilization, Mn allows Gpp(NH)p to stimulate adenylate cyclase by simple Michaelis-Menten kinetics as shown for Mg in particulate and solubilized preparations. 5) The lag associated with inhibition of forskolin-sensitive adenylate cyclase is diminished by Mg but not by Mn. 6) Activation of membrane-bound adenylate cyclase by forskolin is consistent with an action at a single, high-affinity site. After solubilization, activation by forskolin is biphasic, with both high- and low-affinity components becoming apparent. The adenylate cyclase response to forskolin at the low-affinity site is greater with Mn than with Mg.(ABSTRACT TRUNCATED AT 250 WORDS)