Four aspects of the behavior of 3-deoxy-3-fluoro-D-glucose (D-3FDG) were studied. The distribution of label in rat tissues after intravenous administration of [18F]D-3FDG was compared with that seen with labeled 3-deoxy-3-fluoro-L-glucose (L-3FDG). Results were consistent with a larger volume of distribution for the physiological D-isomer coupled with some degree of reabsorption by the kidneys. L-3FDG, but not its D-isomer, was excluded from the brain. D-3FDG competitively inhibited uptake of glucose by isolated perfused rat hearts. The inhibition constant was 12.8 +/- 1.6 mM compared with 6.1 +/- 1.1 mM for 3-O-methyl-D-glucose. Residue curves obtained after bolus administration of [18F]D-3FDG to isolated hearts indicated phosphorylation of the tracer at a lower rate than for 2-deoxy-2-fluoro-D-glucose but with subsequent dephosphorylation at a faster rate. Chromatographic analysis of 18F remaining in tissues after administration of [18F]D-3FDG revealed in addition to free D-3FDG three other peaks. These disappeared after treatment with alkaline phosphatase and were thus assigned as phosphates. The principal metabolite had the same retention time as D-3FDG-6-phosphate prepared with hexokinase. No phosphorylated metabolites were detected in blood. D-3FDG labeled with 18F may be a useful tracer in studies of glucose transport and metabolism.