Extent, time course, and underlying mechanisms of the negative inotropic effect of ryanodine were examined in 22 length-clamped ferret right ventricular papillary muscles paced 12/min at 25° C. After 60 minutes of exposure to 5 μM ryanodine a new steady state was attained with developed forces averaging 10-15% of maximum twitch force. Ryanodine does not pharmacologically excise the sarcoplasmic reticulum (SR) in this preparation. Ryanodine does not appreciably inhibit the ability of the SR to take up Ca2+ as evidenced by the potentiated beats obtained after a short pause that are nearly as large after ryanodine as before. On comparing equipotent beats before and after ryanodine, we found that ryanodine actually increases the rate at which Ca2+ is released during the twitch if the SR Ca2+ stores are equal or similar. The evidence for this conclusion is a larger maximum rate of tension rise and briefer time to peak tension after ryanodine. Since ryanodine increases the time that SR Ca2+ release channels are open and decreases their conductivity, it must follow that the former effect predominates over the latter in our experiments. Ryanodine increases the leakiness of the SR during diastole probably by inhibiting closure of SR Ca2+ release channels. The evidence for this conclusion is as follows: the early peak of the restitution curves after ryanodine, the brevity of the time required for a rested state contraction after ryanodine, and the small amplitude of the steady-state contraction at a rate of 12/min. The SR leaks even in the absence of ryanodine, but if external Ca2+ is so high that Ca2+ loss from the cell is slowed or a Ca2+ leak into the cell through the sarcolemma cancels the SR leak, then the effects of the SR leak are minimized. The evidence for this conclusion is the time required for rested-state contraction to occur or the slope of the descending limb of restitution curve; however, in presence of ryanodine even high external Ca2+ cannot prevent rapid depletion of SR Ca2+ stores. Even though we have presented evidence for a mechanism whereby ryanodine increases the number of open SR Ca2+ release channels in both systole and diastole, we do not mean to imply that most of them stay open in diastole: the SR would leak too fast to accumulate any Ca2+ for the potentiated beat. Thus, probably most channels close after being open a certain length of time, even in the presence of ryanodine.