The isolated working rat heart was adapted for simultaneous determination of glycogen synthesis and degradation using a dual isotope technique. After prelabeling of glycogen with [U-14C]glucose, glycogenolysis was determined continuously from the washout of 14CO2 plus [14C]lactate. Glycogen synthesis was determined during the same period from incorporation of [5-3H]glucose. In the absence of added hormones, hearts were predominantly glycogenolytic (1.5 mumol/min/g, dry weight), and there was simultaneous synthesis (11% of the rate of glycogenolysis). The percentage of glucose taken up by the heart that could traverse the glycogen pool as a consequence of glycogen turnover was minor (5%). Insulin (10 milliunits/ml) predictably stimulated glycogen synthesis (3.6-fold) and nearly abolished glycogenolysis. Addition of glucagon (1 microgram/ml) increased contractile performance and initially stimulated glycogenolysis (3.8-fold) until glycogen was largely depleted. Net tritium incorporation was unaffected by glucagon. Both hormones stimulated glycolytic flux from exogenous glucose (3H2O from [5-3H]glucose) as well as total glycolytic flux (3H2O plus glycogenolysis). The initial stimulation in total glycolytic flux with glucagon was largely from glycogen, explaining the lag in stimulation from exogenous glucose. The relationship between the specific radioactivity and amount of glycogen remaining after different degrees of glycogenolysis suggests that the preference of glycogenolysis for newly synthesized glycogen is only partial.