The quantitative determination of glucose uptake by using 2-deoxy-D-glucose (2-DG) is based on the assumption that 2-deoxyglucose-6-phosphate (2-DG6P) cannot be further metabolized and requires the lumped constant (LC) to equate the kinetic differences in uptake between 2-DG and glucose. We tested whether insulin or epinephrine affect the LC, and quantitated the incorporation of 2-DG6P into glycogen in the isolated working rat heart. Hearts were perfused for 35 min at near physiological workload with Krebs-Henseleit buffer containing glucose (5 mmol/l) plus oleate (0.4 mmol/l, Group 1) with either insulin (1 mU/ml, Group 2), or epinephrine (1 micromol/l, Group 3). In all groups [2-3H] glucose and [U-14C]2-DG (10 microCi each) were present in the perfusate for the first 30 min. In order to estimate the quantitative relationship of glucose and 2-DG uptake and glycogen synthesis from glucose and 2-DG, we perfused hearts with equimolar amounts of glucose and 2-DG (5 mmol each) and either [18F]2-deoxy-2-fluoroglucose plus [2-3H]glucose or [U-14C]glucose plus [1,2-3H]2-DG as tracers. All hearts were freeze-clamped for determination of 2-DG accumulation, glycogen, and tracer activity in glycogen. Glucose and 2-DG uptake were similar in the absence of insulin (LC 1.27+/-0.09). In the presence of insulin, 2-DG underestimated glucose uptake (LC 0.61+/-0.02). Epinephrine did not affect the tracer/tracee ratio (LC 1.31+/-0.09). Incorporation of [U-14C]2-DG into glycogen occurred in all groups (Group 15.38+/-0. 65%, Group 25.72+/-0.59%, Group 32.70+/-0.16% of total tracer uptake.) When equimolar amounts of glucose and 2-DG were present, 2-DG uptake, measured by dynamic assessment of FDG accumulation, significantly decreased over 30 min while glucose uptake remained unchanged. The hearts perfused with [U-14C]glucose and [1,2-3H]2-DG synthesized 39.5+/-7.1 micromol glycogen/g dry/30 min. 2-DG contributed 4.2+/-1.4%. We conclude that insulin and epinephrine have differential effects on the LC, and 2-DG6P is a substrate for glycogen synthesis.