Electro-mechanical and Ca alternans is a known pro-arrhythmic factor. At the cellular level Ca alternans appears as cytosolic Ca transients of alternating amplitude at regular beating frequency. Direct intra-sarcoplasmic reticulum (SR) [Ca] measurements with the low affinity Ca indicator fluo-5N entrapped in the SR revealed that alternans in diastolic SR content are not a prerequisite for cytosolic Ca alternans, and thus SR Ca content is not the sole determinant of alternans. The goal of this study was to determine whether alternans of the kinetics of recovery from inactivation of ryanodine receptors and refractoriness of release represent a key factor underlying cytosolic Ca alternans. Alternans was induced by electrical pacing (1.6 to 2.5 Hz). After Ca alternans was established, pacing was stopped and the occurrence of spontaneous Ca waves and Ca sparks was quantified. The time interval from cessation of stimulation to the appearance of the first Ca waves was significantly shorter and the frequency of Ca sparks was higher after the small Ca transient compared to the large transient. Application of 0.1 mM caffeine or 10 μM isoproterenol rescued Ca alternans and shortened the rest interval until appearance of Ca waves. Photolysis of caged Ca (DM-nitrophen) to produce photolytically triggered Ca release (PTCR) from the SR was used to probe the refractoriness of SR Ca release during alternans. During the decay phase of the Ca transient PTCR was significantly less during the large Ca transient. During the rising phase of the Ca transient PTCR was greater during the large Ca transient, and was capable of inducing a phase reversal of Ca alternans. We conclude that alternating ryanodine receptor inactivation recovery intervals, together with alternations in SR Ca load, represent key determinants of Ca alternans. (VMS and CL contributed equally).