BACKGROUND The glucose tracer analog [18F]2-deoxy-2-fluoro-D-glucose (FDG) is widely used for assessing regional myocardial glucose metabolism in vivo. The reproducibility of this method has recently been questioned because of a discordant affinity of hexokinase for its substrates glucose and 2-deoxyglucose. We therefore compared rates of glucose utilization simultaneously with tissue time-activity curves of FDG uptake before and after changes in the physiological environment of the heart. RESULTS Isolated working rat hearts were perfused for 60 minutes with recirculating Krebs buffer containing glucose (10 mmol/L), FDG (1 microCi/mL), [2-3H]glucose (0.05 microCi/mL), and [U-14C]2-deoxyglucose (2-DG; 0.025 microCi/mL). Myocardial glucose uptake was measured by tracer ([2-3H]glucose) and tracer analog methods (FDG and 2-DG) before and after the addition of either insulin (1 mU/mL), epinephrine (1 mumol/L), lactate (40 mmol/L), or D,L-beta-hydroxybutyrate (40 mmol/L) at 30 minutes of perfusion and after acute changes in cardiac workload. Under steady-state conditions, myocardial rates of glucose utilization as measured by tritiated water (3H2O) production from metabolism of [2-3H]glucose, FDG uptake, and 2-DG retention were linearly related. The addition of competing substrates decreased glucose utilization immediately. The addition of insulin increased the rate of glucose utilization as measured by the glucose tracer but not as measured by the tracer analogs. The ratio of 3H2O release/myocardial FDG uptake increased by 111% after the addition of insulin, by 428% after the addition of lactate, and by 232% after the addition of beta-hydroxybutyrate. Epinephrine increased rates of glucose utilization and contractile performance, whereas there was no increase in glucose uptake with a comparable increase in workload alone. There was no change in the relation between the glucose tracer and the tracer analog either with epinephrine or with acute changes in workload. CONCLUSIONS The uptake and retention of FDG in heart muscle is linearly related to glucose utilization only under steady-state conditions. Addition of insulin or of competing substrates changes the relation between uptake of the glucose tracer and FDG. These observations preclude the determination of absolute rates of myocardial glucose uptake by the tracer analog method under non-steady-state conditions.