Sarcoplasmic reticulum (SR) Ca(2+) leak determines SR Ca(2+) content and, therefore, the amplitude of global Ca(2+) transients in ventricular myocytes. However, it remains unresolved to what extent Ca(2+) leak can be modulated by cytosolic [Ca(2+)] (Ca(2+)). Here, we studied the effects of Ca(2+) on SR Ca(2+) leak in permeabilized rabbit ventricular myocytes. Using confocal microscopy we monitored SR Ca(2+) leak as the change in Ca(2+) (with Fluo-5N) after complete SERCA inhibition with thapsigargin (10 μm). Increasing Ca(2+) from 150 to 250 nM significantly increased SR Ca(2+) leak over the entire range of Ca(2+). This increase was associated with an augmentation of both Ca(2+) spark- and non-spark-mediated Ca(2+) leak. Further increasing Ca(2+) to 350 nM led to rapid Ca](SR) depletion due to the occurrence of Ca(2+) waves. The augmentation of SR Ca(2+) leak by high Ca(2+) was insensitive to inhibition of Ca(2+)-calmodulin-dependent protein kinase II. In contrast, lowering Ca(2+) to 50 nM markedly decreased SR Ca(2+) leak rate and nearly abolished Ca(2+) sparks. When the ryanodine receptor (RyR) was completely inhibited with ruthenium red (50 μM), changes in Ca(2+) between 50 and 350 nM did not produce any significant effect on SR Ca(2+) leak, indicating that Ca(2+) alters SR Ca(2+) leak solely by regulating RyR activity. In summary, Ca(2+) in the range of 50-350 nM has a significant effect on SR Ca(2+) leak rate mainly via direct regulation of RyR activity. As RyR activity depends highly on Ca(2+) and Ca(2+), SR Ca(2+) leak remains relatively constant during the declining phase of the Ca(2+) transient when Ca(2+) and Ca(2+) change in opposite directions.