We tested the hypothesis that glycogen is preferentially oxidized in isolated working rat heart. This was accomplished by measuring the proportion of glycolytic flux (oxidation plus lactate production) specifically from glycogen which is metabolized to lactate, and comparing it to the same proportion determined concurrently from exogenous glucose during stimulation with epinephrine. After prelabeling of glycogen with either 14C or 3H, a dual isotope technique was used to simultaneously trace the disposition of glycogen and exogenous glucose between oxidative and non-oxidative pathways. Immediately after the addition of epinephrine (1 microM), 40-50% of flux from glucose was directed towards lactate. Glycogen, however, did not contribute to lactate, being almost entirely oxidized. Further, glycogen utilization responded promptly to the abrupt increase in contractile performance with epinephrine, during the lag in stimulation of utilization of exogenous glucose, suggesting that glycogen serves as substrate reservoir to buffer rapid increases in demand. Preferential oxidation of glycogen may serve to ensure efficient generation of ATP from a limited supply of endogenous substrate, or as a mechanism to limit lactate accumulation during rapid glycogenolysis.