The difference in histamine receptor subtypes that are involved in the positive inotropic effect of histamine in guinea pig and rabbit ventricular myocardium was analytically characterized. In guinea pig papillary muscles, the positive inotropic effect of histamine was antagonized by cimetidine but not by mepyramine. The converse was true in rabbit papillary muscles. However, histamine evoked a positive inotropic effect through H1- and H2-receptors after blockade of H2- and H1-receptors in guinea pig and rabbit papillary muscles, respectively. Adenylate cyclase was significantly activated by histamine via H2-receptors in guinea pig but not in rabbit myocardial ventricular membranes. Accumulation of [3H]inositol monophosphate in ventricular strips prelabeled with myo-[3H]inositol was increased by histamine via H1-receptors to a similar extent in rabbits and guinea pigs. Radioligand binding experiments with [3H]mepyramine and [3H]tiotidine showed an increased number of H1-receptors and a decreased number of H2-receptors in guinea pig compared with rabbit ventricular myocardium. These results suggest that the positive inotropic effects of histamine are dominated by an H1-receptor-mediated effect in rabbits and by an H2-receptor-mediated one in guinea pig ventricular myocardium, and the positive inotropic effect manifested by one subtype apparently restricts the expression of the positive inotropic effect mediated by the other subtype. This species difference is not due to a difference in densities of the receptor subtypes, but may be partly related to a difference in the extents of coupling of H2-receptors to adenylate cyclase.