Triglyceride turnover in the isolated C57/BL6 mouse heart was measured by dynamic 13C edit-(1)H observe NMR and the rate of fatty acid oxidation was determined by 13C NMR isotopomer analysis. In the presence of a physiological mixture of substrates, energy was produced in the citric acid cycle by oxidation of long-chain fatty acids (18%), ketones (34%), lactate (24%), pyruvate (7%), and other sources (17%). Exogenous fatty acids appeared in the triglyceride pool at 0.24 micromol/g dry wt/min, similar to the rate of oxidation of long-chain fatty acids, 0.16 micromol/g dry wt/min. Isoproterenol decreased the rate of de novo triglyceride synthesis and increased the rate of fatty acid oxidation.