Increased diastolic SR Ca²⁺ leak (J_leak) could depress contractility in heart failure, but there are conflicting reports regarding the J_leak magnitude even in normal, intact myocytes. We have developed a novel approach to measure SR Ca²⁺ leak in intact, isolated ventricular myocytes. After stimulation, myocytes were exposed to 0 Na⁺, 0 Ca²⁺ solution ±1 mmol/L tetracaine (to block resting leak). Total cell [Ca²⁺] does not change under these conditions with Na⁺-Ca²⁺ exchange inhibited. Resting [Ca²⁺]_i declined 25% after tetracaine addition (126±6 versus 94±6 nmol/L; P<0.05). At the same time, SR [Ca²⁺] ([Ca²⁺]_SRT) increased 20% (93±8 versus 108±6 μmol/L). From this Ca²⁺ shift, we calculate J_leak to be 12 μmol/L per second or 30% of the SR diastolic efflux. The remaining 70% is SR pump unidirectional reverse flux (backflux). The sum of these Ca²⁺ effluxes is counterbalanced by unidirectional forward Ca²⁺ pump flux. J_leak also increased nonlinearly with [Ca²⁺]_SRT with a steeper increase at higher load. We conclude that J_leak is 4 to 15 μmol/L cytosol per second at physiological [Ca²⁺]_SRT. The data suggest that the leak is steeply [Ca²⁺]_SRT-dependent, perhaps because of increased [Ca²⁺]_i sensitivity of the ryanodine receptor at higher [Ca²⁺]_SRT. Key factors that determine [Ca²⁺]_SRT in intact ventricular myocytes include (1) the thermodynamically limited Ca²⁺ gradient that the SR can develop (which depends on forward flux and backflux through the SR Ca²⁺ ATPase) and (2) diastolic SR Ca²⁺ leak (ryanodine receptor mediated).