Skinned fiber bundles from papillary muscle of rabbits were used to study the effects of ryanodine (1) on direct Ca2+ activation of the contractile proteins, and (2) on direct Ca2+ uptake and release from the sarcoplasmic reticulum (SR). Caffeine (25 mM) was used to release Ca2+ from the SR and to generate a tension transient. Each tension transient occurred after sequential immersion of the fiber bundles into five solutions: loading (uptake phase, [U]) and releasing (release phase, [R]). The height of free Ca2+-activated tension development of the contractile proteins, and the area of the tension transient generated by caffeine were assessed. (1) The direct free Ca2+-activated tension development of the contractile proteins was not significantly affected by ryanodine up to 0.1 mM, either at the submaximal or maximal free Ca2+ concentrations. (2) Ryanodine (1 nM-1 microM), in U, R, or in U and R, did not significantly change the immediate caffeine-induced tension transients. In the same preparation after ryanodine treatments, the second control caffeine-induced tension transients (C2, no ryanodine) were decreased in a dose-dependent manner (IC50 = 50 nM, 10 nM, 10 nM for R, U, and U and R, respectively). The depression caused by ryanodine on the SR was "activity"-dependent and not readily reversible. Total calcium content in the SR of C2 was not significantly changed by small quantities of ryanodine (less than 0.1 microM) and was decreased with greater amounts of ryanodine (greater than or equal to 0.1 microM). Thus, at low concentrations of ryanodine, the negative inotropic action is due to decrease Ca2+ release from the SR; at high concentration of ryanodine, it is due to decrease in calcium accumulation in the SR.