Since it has been known that ryanodine has a potent negative inotropic effect on the cardiac muscle contractility (Jenden and Fairhurst, 1968), ryanodine has been a subject of intensive research (Frank and Sleator, 1975; Jones et al, 1978; Sutko et al, 1985). However, the underlying mechanism for the ryanodine dependent negative inotropic effect is still uncertain. In this study, the effects of ryanodine on the generation and relaxation of contracture due to Na-withdrawal and on the force-frequency relationship of heart muscles isolated from rats and guinea pigs were measured in an effort to understand the underlying mechanism of the ryanodine-induced negative inotropy. Results are summerized as follows: 1 ) Ryanodine significantly reduced the contractility of heart muscles produced at low frequency of stimulation, but showed a little effect on the contractility at high frequency stimulation. 2) Ryanodine, at the concentrations ranging from 10^-6 M to 10^-8 M, had no significant effect on the Na-dependent relaxation of Na-withdrawl contracture. 3) Ryandoine significantly reduced the amplitude of the Na-withdrawl contracture, and this inhibitory effect was reinforced by procaine, antiagonized by caffeine and high potassium. From these results, it may be concluded that the negative inotropic effect of ryanodine is mainly due to an inhibition of calcium release from sarcoplasmic reticulum.