2-Deoxy-2-[¹⁸F]fluoro-D-glucose (FDG) may be used to predict glucose kinetics when the factor relating differences in transport and phosphorylation between compounds remains constant ("lumped constant"). It is not clear whether hyperemia alters that factor. In anesthetized swine, myocardial FDG uptake was estimated by positron emission tomography, during an intracoronary infusion of either adenosine, ATP, or bradykinin (40 µg · kg¹ · min¹, 40 µg · kg¹ · min¹, and 2 nmol · kg¹ · min¹, respectively; n = 6 for all groups). In controls during normal perfusion (n = 6), FDG uptake was 0.78 ± 0.32 µmol · g¹ · min¹, whereas glucose uptake by Fick was 0.71 ± 0.25 µmol · g¹ · min¹ (r = 0.73; P < 0.05). Adenosine increased blood flow from 1.29 ± 0.43 to 4.80 ± 2.19 ml · g¹ · min¹ (P < 0.05) and glucose uptake from 1.16 ± 1.10 to 3.35 ± 2.12 µmol · g¹ · min¹ (P < 0.05), whereas FDG uptake in the hyperemic region was lower than remote regions (0.46 ± 0.29 and 0.95 ± 0.55 µmol · g¹ · min¹, respectively; P < 0.05). In the ATP and bradykinin groups, blood flow increased four- and twofold, respectively, with no net change in glucose uptake. FDG uptake in the hyperemic region was also significantly lower than remote regions. For all animals, the ratio of blood flow in the hyperemic region relative to remote region was inversely proportional to the ratio of FDG uptake in the same regions (r²=0.73; P < 0.001). Because nitric oxide elaboration during hyperemia could potentially alter substrate preference and FDG kinetics, six additional swine were studied during maximal adenosine before and after intracoronary N^G-monomethyl-L-arginine (1.5 mg/kg). Inhibition of nitric oxide had no effect on either regional myocardial substrate uptake or FDG accumulation. In conclusion, hyperemia decreased regional myocardial FDG uptake relative to normally perfused regions and this effect on the lumped constant was independent of nitric oxide.