To examine the use of 2-deoxy-2-[18F]fluoro-D-glucose (2-FDG) as a glucose analog for measuring glucose utilization rate in myocardium, dual-tracer kinetic experiments with 2-FDG and 2-[3H]glucose were performed in the perfused, isolated rabbit interventricular septum to measure simultaneously the transport and phosphorylation rates of glucose and 2-FDG. Results of the present study indicated that, in the septum, the transport rate constants of 2-FDG and glucose were similar in magnitude, the phosphorylation rate constant for 2-FDG was about 60% of that of glucose, hypoxia caused an increase in phosphorylation rates of glucose and 2-FDG without affecting transport.