To assess kinetic changes of myocardial glucose metabolism after physiological interventions, we perfused isolated working rat hearts with glucose and 2-[18F]fluoro-2-deoxy-D-glucose (2-FDG). Tissue uptake of 2-FDG and the input function were measured on-line by external detection. The fractional rate of 2-FDG phosphorylation was determined by graphical analysis of time-activity curves. The steady-state uptake of 2-FDG was linear with time, and the tracer was retained predominantly in its phosphorylated form. Tissue accumulation of 2-FDG decreased with a reduction in work load and with the addition of competing substrates. Insulin caused a significant increase in 2-FDG accumulation in hearts from fasted but not from fed animals. We conclude that in the isolated working rat heart there is rapid adjustment of exogenous substrate utilization and that most interventions known to alter glucose metabolism induce parallel changes in 2-FDG uptake. Qualitative differences in the in vitro response to insulin may be affected by the presence of either endogenous insulin or glycogen.