On perfusing the isolated rat heart with a medium containing Ca45, mitochondrial fraction was found to contain more radioactivity than sarcoplasmic reticulum. Not only the calcium contents of mitochondria were higher but also the specific activity of calcium pool of mitochondria was greater than that of the sarcoplasmic reticulum. The in vitro studies revealed that during 10 min of incubation mitochondria accumulated more calcium and hydrolyzed less ATP in comparison to the sarcoplasmic reticulum. Calcium accumulation and ATP hydrolysis by mitochondria were markedly inhibited by 5 mMazide. On the other hand, azide had a slight inhibitory action on the ATPase activity of sarcoplasmic reticulum but did not influence its ability to bind calcium. On the basis of 1 min calcium binding values, total mitochondria and sarcoplasmic reticulum present in the heart are considered to accumulate 0.395 and 0.085 µmol of calcium/g of heart. It is suggested that free calcium concentration in the heart is determined mainly by the activities of mitochondria, sarcoplasmic reticulum, contractile proteins and movements across the cell membrane.