Ruthenium Red (RuR) blocks conduction of Ca²⁺ through single cardiac ryanodine receptor (RyR2). Studies on isolated channels indicate that RuR acts by decreasing open probability without changing single channel conductance(Lukyanenko et al., 2000; Xu et al., 1999). This study examines the time course of RuR action on Ca²⁺ efflux from the sarcoplasmic reticulum (SR) of rabbit ventricular myocytes and compares the timecourse of the block with that the effects on Ca²⁺ sparks activity. Rabbit cardiac myocytes were enzymatically isolated from New Zealand White male rabbits killed with an intraveneous injection of sodium pentobarbitone (100 mg kg^-1). Ca²⁺ fluxes were studied on populations cells using a cuvette-based system and Ca²⁺ sparks were monitored in single cells using confocal microscopy. For the cuvette studies, aliquots of 2×10⁶ cells were permeabilised by brief exposure to 0.01 mg β-escin and the cells were placed in a cuvette containing a mock intracellular solution (1.2ml) of the following composition (mM): 0.05 EGTA, 25 Hepes, 0.9 Mg²⁺, 100 KCl, 0.01 Na⁺, pH 7.0 (20-22°C). Free [Ca²⁺] was monitored with the addition of 10µM Fura2 (free acid). After addition of 30 mmoles of Ca²⁺, 20mM caffeine was added to induce a large Ca²⁺ leak. Under these conditions the [Ca²⁺] within the cuvette increased to 540±23nM (n=4). Addition of 4μM RuR caused a rapid reduction in Fura-2 fluorescence reflecting RuR block of RyR2. The Fura-2 signal reached a steady state after ~10mins (mean value 238±35 nM, n=4). The block was evident immediately with a decrease to 82±4% of control occurring in the 1st minute and reaching 37±6% of control by 4mins. In parallel confocal experiments, β-escin-treated cardiomyocytes were perfused with mock intracellular solutions containing ~600nM Ca²⁺ and 300 mM EGTA. On exposure to 4μM RuR, spark frequency was not significantly different from control after 2 mins, while slightly lower (84±13%) after 3 mins, and reduced to 54±13% of control value after 5 mins. Remarkably, spark width, duration and amplitude remained unchanged from control values. This suggests that RyR2-mediated Ca²⁺ flux is more sensitive to RuR than Ca²⁺ sparks. The reason for this discrepancy is unknown, one possibility is that there may be a significant population of RyR’s that are not associated with the diadic cleft and do not participate in Ca²⁺ sparks. These RyR2 may be more sensitive to block by RuR.