Diastolic waves of Ca²⁺ release have been shown to activate delayed afterdepolarizations as well as some cardiac arrhythmias. The aim of this study was to investigate whether increasing ryanodine receptor open probability alone or in the presence of β-adrenergic stimulation produces diastolic Ca release from the sarcoplasmic reticulum (SR). When voltage-clamped rat ventricular myocytes were exposed to caffeine (0.5 to 1.0 mmol), diastolic Ca²⁺ release was seen to accompany the first few stimuli but was never observed in the steady state. We attribute the initial phase of diastolic Ca²⁺ release to a decrease in the threshold SR Ca²⁺ content required to activate Ca²⁺ waves and its subsequent disappearance to a decrease of SR content below this threshold. Application of isoproterenol (1 μmol/L) increased the amplitude of the systolic Ca²⁺ transient and also the SR Ca²⁺ content but did not usually produce diastolic Ca²⁺ release. Subsequent addition of caffeine, however, resulted in diastolic Ca²⁺ release. We estimated the time course of recovery of SR Ca²⁺ content following recovery from emptying with a high (10 mmol/L) concentration of caffeine. Diastolic Ca²⁺ release recommenced only when SR content had increased back to its final level. We conclude that increasing ryanodine receptor open probability alone does not produce arrhythmogenic diastolic Ca²⁺ release because of the accompanying decrease of SR Ca²⁺ content. β-Adrenergic stimulation increases SR content and thereby allows the increased ryanodine receptor open probability to produce diastolic Ca²⁺ release. The implications of these results for arrhythmias associated with abnormal ryanodine receptors are discussed.