Ryanodine has different effects on the contractility of rat and guinea pig ventricular muscle. Thus we investigated the effect of ryanodine on the intracellular Ca²⁺ and Na⁺ activities of the rat and guinea pig ventricular myocytes with two specific aims; whether there are any differences in intracellular Na⁺ activities between rat and guinea pig ventricular muscle cells, and if any, how the differences in intracellular Na⁺ activities are related to the effect of Na⁺-Ca²⁺ exchange on the action potential configuration and excitation-contraction coupling of the rat and guinea pig ventricular myocytes. Ryanodine (10^-7 M) diminished the slow repolarization phase of the rat ventricular action potential while the duration of the rapid repolarization phase increased. Ryanodine (10^-7 M) significantly increased the plateau of the action potential. At the steady state of 0.2 cps, intracellular Na⁺ activities (aⁱ_Na) of the rat and guinea pig ventricular myocytes were 8.7 ± 5.2 mM (n = 16, 4 rats) and 10.0 ± 4.1 mM (n = 25, 7 guinea pigs) respectively, but there were no statistically significant differences. The contractility of the rat ventricular muscle nearly disappeared due to ryanodine (10^-7 M) with little changes in aⁱNa. Monensin (10 mM) not only increased the resting tension but also remarkably increased aⁱNa from 2.0 mM to 20 mM. Ryanodine (10^-7 M) continuously decreased aⁱNa of the guinea pig ventricular muscle after the contraction ceased to decrease. Monensin increased the contractility as well as aⁱNa. These results suggest that the contractility of rat and guinea pig ventricular myocytes is determined by the change in the action of the Na⁺-Ca²⁺ exchange mechanism depending upon the plateau of action potential and the intracellular Na⁺ and Ca²⁺ activities. So ryanodine could decreases the contractility via its effect on Na⁺-Ca²⁺ exchange transport which could be one of possible mechanisms of negative inotropism by ryanodine.