We postulated that glycogen is a significant energy substrate compared with fatty acids and glucose in response to adrenergic stimulation of working rat hearts. Oxidation rates were determined at 1-min intervals by release of³H₂O from [9,10-³H]oleate (0.4 mM, 1% albumin) and¹⁴CO₂from exogenous [U-¹⁴C]glucose (5 mM) or, by a pulse-chase method, from [¹⁴C]glycogen. We estimated the ¹⁴C enrichment of glycogen metabolized at each time point to determine true rates of glycogen use. Based on the pattern of glycogen enrichment over time, glycogenolysis did not exhibit a high degree of preference for newly synthesized glycogen. Epinephrine (1 μM) increased contractile performance 86% but did not stimulate oleate oxidation. The increased energy demand was supplied by carbohydrates, initially by a burst of glycogenolysis (contributing 35% to total ATP synthesis for 5 min) and followed by delayed increase in the use of exogenous glucose (eventually contributing 29% to ATP synthesis). On the basis of the release of¹⁴CO₂and [¹⁴C]lactate specifically from glucose or glycogen, we found that a larger portion of glycogen was oxidized compared with exogenous glucose, augmenting the yield of ATP from glycogen. Thus the heart responds to an acute increase in energy demand by selective oxidation of glycogen.