Ryanodine and caffeine have the ability to diminish sarcoplasmic reticulum (SR) calcium release in cardiac muscle cells. To determine whether these agents also share a common mechanism of action, we compared their effects on rat papillary muscles using two different experimental approaches. First, using the protocol of Jundt et al. (19), in which quiescent rat papillary muscles were exposed to sodium-free solutions, we found that 1 microM ryanodine decreased resting force, phosphorylase alpha activity, and the scattered light intensity fluctuations (SLIF) due to calcium-dependent myofilament interactions. In contrast, 10-20 mM caffeine increased both resting force and phosphorylase alpha levels and initially increased then decreased SLIF to below detectable levels. In a second series of experiments, contractures were elicited by exposing rat papillary muscles to 125 mM KCl. Pretreatment with ryanodine (1 microM) or caffeine (10 mM) abolished the initial phasic component of this response, while increasing the subsequent tonic component. These effects were different from those of isoproterenol, which decreased tonic contracture force. The depression of twitch force produced by ryanodine developed more rapidly in the presence of 125 mM KCl than in normal buffer, suggesting that the negative inotropic effects of this agent may, in part, depend on membrane depolarization. The results of these experiments suggest that ryanodine and caffeine affect SR calcium release through different mechanisms of action. Ryanodine appears to decrease, while caffeine initially increases, cytoplasmic calcium. Once these effects have occurred, the alterations of SR function produced by both agents can similarly alter other inotropic responses.