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+)](i)). Here, we studied the effects of [Ca(2+)](i) 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+)](SR) (with Fluo-5N) after complete SERCA inhibition with thapsigargin (10 μm). Increasing [Ca(2+)](i) from 150 to 250 nM significantly increased SR Ca(2+) leak over the entire range of [Ca(2+)](SR). This increase was associated with an augmentation of both Ca(2+) spark- and non-spark-mediated Ca(2+) leak. Further increasing [Ca(2+)](i) to 350 nM led to rapid [Ca](2+)](SR) depletion due to the occurrence of Ca(2+) waves. The augmentation of SR Ca(2+) leak by high [Ca(2+)](i) was insensitive to inhibition of Ca(2+)-calmodulin-dependent protein kinase II. In contrast, lowering [Ca(2+)](i) 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+)](i) between 50 and 350 nM did not produce any significant effect on SR Ca(2+) leak, indicating that [Ca(2+)](i) alters SR Ca(2+) leak solely by regulating RyR activity. In summary, [Ca(2+)](i) 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+)](i) and [Ca(2+)](SR), SR Ca(2+) leak remains relatively constant during the declining phase of the Ca(2+) transient when [Ca(2+)](SR) and [Ca(2+)](i) change in opposite directions.