Myocardial compartmentation of calcium was investigated in the arterially perfused rabbit interventricular septum under conditions of augmented calcium uptake. Reduction of perfusate sodium concentration (100-36 mM [Na]0) produced the expected increased active force development and an increased myocardial calcium content that was inversely proportional to [Na]0. Caffeine was used to inhibit calcium uptake by the sarcoplasmic reticulum (SR) and to stimulate SR calcium release. The diastolic tension response to caffeine was also inversely proportional to [Na]0: at [Na]0 of 139 mM, 10 mM caffeine increased diastolic tension by 20%; whereas at 36 mM [Na]0 diastolic tension increased by 205%. The increase in diastolic tension in response to caffeine was considered a reflection of increased cytosolic calcium. The increase in diastolic tension with caffeine required that reduced [Na]0 be present at the time caffeine was administered. Caffeine sensitivity (measured by an increase in diastolic tension) and active force development declined to control levels within 3 minutes after the end of a 40 minute period of low [Na]0 perfusion despite the presence of an additional 1 mmole calcium per kg dry wt in the muscle at the 3 minute mark when caffeine was added. The results indicate that low [Na]0 perfusion induces an increment in myocardial calcium content, a major fraction of which is neither related directly to contractility nor involved in the response to caffeine.