To test the hypothesis that ryanodine inhibits the release of contractile Ca2+from intracellular stores, we compared the contractile responses by rabbit, cat, dog, and rat papillary muscles to ryanodine. Results of cumulative ryanodine concentration (10−9to 10−4M) response studies indicate the following order of sensitivity to ryanodine: rat > dog = cat > rabbit which mimics the relative dependence of these species on intracellular Ca2+for force development. In the presence of 2.5 mM [Ca2+]$, cumulative additions of ryanodine or a single exposure to 10−4M concentration produced biphasic contractile responses in rabbit, cat, and dog, but not rat ventricular muscle. The elevation of [Ca2+]oto 5 RIM either antagonized the expression of ryanodine's negative inotropic effect or promoted the positive effect of this agent in all species tested. Ryanodine did produce a biphasic change in contractility in the presence of 2.5 mM [Ca2+]oin K+-depolarized, isoproterenol-restored rat papillary muscles. In addition, prior exposure of rat myocardium to ryanodine (2.5 mM [Ca2+]o) was similar to a decreased [Ca2+]oin that it permitted inotropic agents, such as increased stimulation rates, hyperos- molality, and ouabain to produce positive contractile responses from this tissue. In contrast, the positive response by rat cardiac muscle to paired electrical stimulation is prevented by ryanodine. Ryanodine also accelerated the rest-decay of force development in rat myocardium, suggesting that it increased the rate of loss of calcium from contractile-dependent Ca2+stores. The results are consistent with ryanodine effecting a decreased availability of intracellular contractile Ca2+, perhaps through a diminishment of its release. Circ Res 46: 332-343, 1980