Cardiac alternans are thought to be a precursor to life-threatening arrhythmias. Previous studies suggested that alterations in sarcoplasmic reticulum (SR) Ca2+ content are either causative or not associated with myocyte Ca2+ alternans. However, those studies used indirect measures of SR Ca2+. Here we used direct continuous measurement of intra-SR free [Ca2+] ([Ca2+]SR) (using Fluo5N) during frequency-dependent Ca2+ alternans in rabbit ventricular myocytes. We tested the hypothesis that alternating [Ca2+]SR is required for Ca2+ alternans. Amplitudes of [Ca2+]SR depletions alternated in phase with cytosolic Ca2+ transients and contractions. Some cells showed clear alternation in diastolic [Ca2+]SR during alternans, with higher [Ca2+]SR before the larger SR Ca2+ releases. However, the extent of SR Ca2+ release during the small beats was smaller than expected for the modest decrease in [Ca2+]SR. In other cells, clear Ca2+ alternans was observed without alternations in diastolic [Ca2+]SR. Additionally, alternating cells were observed, in which diastolic [Ca2+]SR fluctuations occurred interspersed by depletions in which the amplitude was unrelated to the preceding diastolic [Ca2+]SR. In all forms of alternans, the SR Ca2+ release rate was higher during large depletions than during small depletions. Although [Ca2+]SR exerts major influence on SR Ca2+ release, alternations in [Ca2+](SR) are not required for Ca2+ alternans to occur. Rather, it seems likely that some other factor, such as ryanodine receptor availability after a prior beat (eg, recovery from inactivation), is of greater importance in initiating frequency-induced Ca2+ alternans. However, once such a weak SR Ca2+ release occurs, it can result in increased [Ca2+]SR and further enhance SR Ca2+ release at the next beat. In this way, diastolic [Ca2+]SR alternans can enhance frequency-induced Ca2+ alternans, even if they initiate by other means.