In cardiac muscle, excitation-contraction (E-C) coupling is determined by the ability of the sarcoplasmic reticulum (SR) to store and release Ca2+. It has been hypothesized that the Ca2+ sequestration and release mechanisms might be functionally linked to optimize the E-C coupling process. To explore the relationships between the loading status of the SR and functional state of the Ca2+ release mechanism, we examined the effects of changes in SR Ca2+ content on spontaneous Ca2+ sparks in saponin-permeabilized and patch-clamped rat ventricular myocytes. SR Ca2+ content was manipulated by pharmacologically altering the capacities of either Ca2+ uptake or leak. Ca2+ sparks were recorded using a confocal microscope and Fluo-3 and were quantified considering missed events. SR Ca2+ content was assessed by application of caffeine. Exposure of permeabilized cells to anti-phospholamban antibodies elevated the SR Ca2+ content and increased the frequency of sparks. Suppression of the SR Ca2+ pump by thapsigargin lowered [Ca2+]SR and reduced the frequency of sparks. The ryanodine receptor (RyR) blockers tetracaine and Mg2+ transiently suppressed the frequency of sparks. Upon washout of the drugs, sparking activity transiently overshot control levels. Low doses of caffeine transiently potentiated sparking activity upon application and transiently depressed the sparks upon removal. In patch-clamped cardiac myocytes, exposure to caffeine produced only a transient increase in the probability of sparks induced by depolarization. We interpret these results in terms of a novel dynamic control scheme for SR Ca2+ cycling. A central element of this scheme is a luminal Ca2+ sensor that links the functional activity of RyRs to the loading state of the SR, allowing cells to auto-regulate the size and functional state of their SR Ca2+ pool. These results are important for understanding the regulation of intracellular Ca2+ release and contractility in cardiac muscle.