The effects of changes in heart rate and contractile force due to electrical stimulation, alteration of the temperature of the perfusion medium, and varying degrees of stretch tension on the subcellular distribution of calcium in the isolated perfused rat heart were studied. Increasing the frequency of stimulation from 80 to 320 pulses/min decreased myocardial contractility without appreciable changes in the levels of calcium in the tissue homogenate and heavy microsomal (8000 – 40 000 × g) fraction. On the other hand, mitochondrial (800–8000 × g) calcium content was greater in hearts stimulated at 120 pulses/min than in hearts stimulated at 80 pulses/min; further increase in the frequency of stimulation resulted in a decrease in the level of mitochondrial calcium. Increasing the temperature of the perfusion medium from 25 to 37 °C increased the heart rate and decreased the contractile force without any changes in the levels of calcium in the homogenate or subcellular fractions. Increasing the stretch tension on the hearts from 0 to 5 g increased the developed contractile force and the level of heavy microsomal calcium without changing the heart rate or the levels of calcium in the homogenate and mitochondria. The results do not reveal any cause–effect relationship between changes in heart function and subcellular distribution of calcium.