In this study, Ca 2+ release due to spontaneous Ca 2+ waves was measured both from inside the sarcoplasmic reticulum (SR) and from the cytosol of rabbit cardiomyocytes. These measurements utilized Fluo5N-AM for intra-SR Ca 2+ from intact cells and Fluo5F in the cytosol of permeabilized cells. Restricted subcellular volumes were resolved with the use of laser-scanning confocal microscopy. Local Ca 2+ signals during spontaneous Ca 2+ release were compared with those induced by rapid caffeine application. The free cytoplasmic [Ca 2+] increase during a Ca 2+ wave was 98.1% ± 0.3% of that observed during caffeine application. Conversion to total Ca 2+ release suggested that Ca 2+ release from a Ca 2+ wave was not significantly different from that released during caffeine application (104% ± 6%). In contrast, the maximum decrease in intra-SR Fluo-5N fluorescence during a Ca 2+ wave was 82.5% ± 2.6% of that observed during caffeine application. Assuming a maximum free [Ca 2+] of 1.1 mM, this translates to a 96.2% ± 0.8% change in intra-SR free [Ca 2+] and a 91.7% ± 1.6% depletion of the total Ca 2 +. This equates to a minimum intra-SR free Ca 2+ of 46 ± 7 μM during a Ca 2+ wave. Reduction of RyR2 Ca 2+ sensitivity by tetracaine (50 μM) reduced the spontaneous Ca 2+ release frequency while increasing the Ca 2+ wave amplitude. This did not significantly change the total depletion of the SR (94.5% ± 1.1%). The calculated minimum [Ca 2+] during these Ca 2+ waves (87 ± 19 μM) was significantly higher than control ( p < 0.05). A computational model incorporating this level of Ca 2+ depletion during a Ca 2+ wave mimicked the transient and sustained effects of tetracaine on spontaneous Ca 2+ release. In conclusion, spontaneous Ca 2+ release results in substantial but not complete local Ca 2+ depletion of the SR. Furthermore, measurements suggest that Ca 2+ release terminates when luminal [Ca 2+] reaches ∼50 μM.