Rat hearts were perfused for 40 min with aerobic medium containing different concentrations of calcium (0-5 mM) and their abilities to take up and oxidize glucose, and to produce lactate and glycerol were examined in addition to measuring glycogen, lipids, cyclic AMP, and high energy phosphate stores. Increasing the concentration of calcium was found to decrease myocardial glycogen but increase glucose uptake, glucose oxidation, and lactate release. A decrease in myocardial triglycerides and an increase in free fatty acid contents as well as glycerol release without any changes in cholesterol and phospholipid contents were observed upon increasing the concentration of external calcium. In comparison with the hearts perfused with Ca2+-free medium, the levels of creatine phosphate and ATP were lower and that of ADP higher in hearts perfused with medium containing 5 mM calcium. No differences in AMP and cyclic AMP contents were seen among hearts perfused with different concentrations of calcium. The contractile activity initially increased upon increasing the concentration of calcium from 1.25 to 5 mM and then declined towards the control level. The hearts were unable to generate contractile force in the absence of calcium, whereas the contractile force decreased and then began to recover upon perfusing the hearts with 0.31 mM calcium. These results indicate that elevated levels of intracellular calcium stimulate glycogenolytic, glycolytic, and lipolytic processes in myocardium directly.