Histamine has positive inotropic and chronotropic effects on the heart which are not abolished by beta adrenergic-blocking agents. Since the positive inotropic and chronotropic effects of other hormones on the heart are thought to be mediated by cyclic 3′,5′-AMP, we examined the effect of histamine on adenyl cyclase in particulate preparations of guinea pig, cat, and human myocardium. Histamine at the peak of its dose-response curve, 3 × 10-4moles/liter, produced approximately a 300% increase in cyclic 3′,5′-AMP accumulation in the guinea pig, 60% in the cat, and 90% in the human heart particles. Half-maximal activity for the histamine mediated activation of adenyl cyclase in the guinea pig was 9 × 10-6moles/liter, almost identical with that observed for norepinephrine in the same preparation. DL-Propranolol, 1 × 10-5moles/liter, did not abolish the activation of adenyl cyclase produced by histamine but did abolish the activation produced by norepinephrine. In contrast, diphenhydramine hydrochloride, Benadryl, 8 × 10-5moles/liter, abolished the activation of adenyl cyclase by histamine but not that produced by norepinephrine. These data suggest that there are at least two receptor sites in guinea pig heart mediating the activation of adenyl cyclase, one responsive to histamine, the other to norepinephrine. In addition, combined maximal doses of histamine and norepinephrine produced completely additive effects on the activation of adenyl cyclase, which suggests that at least two separate adenyl cyclase systems are present in the heart, each responsive to one of these hormones. However, definitive proof would require physical separation of the two enzymes.