Histamine stimulates adenylate cyclase activity in a washed membrane preparation from guinea-pig ventricle. Marked synergistic effects are observed with histamine and GTP. In the absence of GTP, the degree of stimulation of the enzyme by histamine is slight and occurs only in the presence of relatively high concentrations of ATP suggesting that ATP, or contaminating GTP in commercial preparations of ATP, may partially satisfy the guanylnucleotide requirement. The GTP analog, GppNHp, strongly and irreversibly activates the cardiac enzyme. Preincubation studies, in which the membranes are treated with GppNHp alone or in combination with histamine followed by estensive washing, indicate that histamine markedly increases the rate of activation of the enzyme by the guanylnucleotide. It is suggested that the mechanism of action of histamine on adenylate cyclase involves a facilitation of the interaction of guanylnucleotides with the regulatory site of the enzyme. The relative activities for stimulation of adenylate cyclase of a series of histamine analogs correlate quite well with the activities of these derivatives on four H2-receptor systems, including atrial rate and ventricular contractility and do not correlate with the activities on H1-receptors. The H2-receptor antagonists, burimamide and metiamide, competitively inhibit hitamine-stimulated adenylate cyclase and the dissociation constants for these antagonists on the enzyme agree with the pharmacological data on the H2-receptors in the atria and ventricles. Our results suggest that histamine-stimulated cardiac adenylate cyclase can be classified inotorpic and chronotropic effects of histamine on the intact heart.