1. The effects of glucose, pyruvate and lactate on basal metabolism and on contraction-related energy expenditure of thin trabeculae isolated from guinea-pig heart were studied using a microcalorimetric technique. 2. Resting heat rates of cardiac ventricular muscle measured in the presence of substrate-free solution (56 +/- 20 mW (g dry weight)-1), 10 mM-lactate (54 +/- 12 mW (g dry weight)-1) and 10 mM-glucose (63 +/- 24 mW (g dry weight)-1) did not differ significantly. Increasing the external glucose concentration (up to 100 mM) and/or adding insulin (up to 80 units l-1) had virtually no effect on the measured resting heat rate. 3. With 10 mM-pyruvate as substrate resting heat rate was substantially larger (106 +/- 40 mW (g dry weight)-1) than with glucose, lactate or substrate-free solution. The concentrations of pyruvate producing a half-maximal increase in resting heat rate as compared to substrate-free solution ranged between 0.4 and 1.2 mM. 4. In order to test whether the development of an anoxic core contributed to the substrate dependence of resting heat production the critical PO2 (i.e. the PO2 that produced a just-noticeable decrease in heat rate) was determined in cylindrical preparations of various diameters. It was found that none of the preparations had an anoxic core at rest in a solution equilibrated with 100% oxygen. 5. From the dependence of the critical PO2 on the diameter of the preparation the diffusion coefficient of oxygen through cardiac muscle was calculated using a modification of Hill's equation (Hill, 1928). The O2 diffusion coefficient was found to be 1.09 X 10(-5) cm2 s-1. 6. Contraction-related heat production was also found to be dependent on the substrate used. In the presence of 10 mM-pyruvate it was about 60% larger than in the presence of 10 mM-glucose, 10 mM-lactate or with substrate-free solution. 7. Isometric force of contraction showed the same substrate dependence as contraction-related heat production and increased with a similar time course during repetitive stimulation. 8. The possible mechanisms underlying the substrate dependence of myocardial energy metabolism are discussed. It is suggested that the increased energy expenditure observed in the presence of pyruvate may be related to a decrease in intracellular phosphate and/or to an increase in intracellular pH.