When ventricular myocytes are overloaded with Ca2+, spontaneous waves of Ca2+-induced Ca2+-release are triggered from the sarcoplasmic reticulum (SR). These waves can result in arrhythmias. Previous work has suggested waves occur when the SR Ca2+ content reaches a threshold level, with the time between waves depending on the rate that the SR refills with Ca2+. It is, however, also possible that the recovery of the SR Ca2+ release channel (Ryanodine Receptor, RyR) from inactivation also plays a role in wave initiation. Most previous work has used indirect methods to measure SR Ca2+ content, and therefore, the mechanisms underlying wave initiation have not been clarified. In order to investigate this issue, we measured free intra-SR and cytosolic Ca2+ simultaneously in canine ventricular myocytes displaying spontaneous Ca2+ waves. Canine myocytes were isolated and dual loaded with Mag-Fura-2AM/Fluo-3AM. Waves were induced via two methods; in some experiments, external Ca2+ concentration was increased up to 15 mM, and in other experiments the cells were voltage clamped in the perforated patch configuration with an elevated intracellular Na+ (15mM). In both cases, wave frequency was altered by changing external Ca2+ concentration. We find that the cytoplasmic Ca2+ wave is associated with a depletion of SR free Ca2+, and recovery of cytoplasmic Ca2+ is associated with a rapid partial recovery of SR free Ca2+. The remainder of the recovery of SR free Ca2+ occurs more slowly. Elevation of external Ca2+ concentration accelerates this slow phase of recovery. When SR free Ca2+ has recovered to the pre-wave level, another wave is initiated, suggesting that a threshold level has been attained. The slow phase of restoration of SR free Ca2+ is associated with either a small decrease of cytoplasmic Ca2+ or no measurable change. Our results confirm a threshold-mediated mechanism for induction of spontaneous waves, with little evidence of a role for RyR refractoriness.