The purpose of the present study was to evaluate a novel approach for determining skeletal muscle-specific glucose flux using radioactive stereoisomers and the microdialysis technique. Microdialysis probes were inserted into the vastus lateralis muscle of human subjects and perfused (4 µl/min) with a Ringer solution containing small amounts of radioactive D- and L-glucose as the internal reference markers for determining probe recovery as well as varying concentrations of insulin (0-10 µM). The rationale behind this approach was that both stereoisomers would be equally affected by the factors that determine probe recovery, with the exception of L-glucose, which is nonmetabolizable and would not be influenced by tissue uptake. Therefore, any differences in the probe recovery ratios between the D- and L-stereoisomers represent changes in skeletal muscle glucose uptake directly at the tissue level. There were no differences in probe recovery between the D- (42.3 ± 3.5%) and L- (41.2 ± 3.5) stereoisomers during the control period (no insulin), which resulted in a D/L ratio of 1.04 ± 0.03. However, during insulin perfusion (1 µM), The D/L ratio increased to 1.62 ± 0.08 and 1.58 ± 0.07 (P < 0.05) during the two collection (0-15 and 15-30 min) periods, respectively. This was accomplished solely by an increase (P < 0.05) in D-glucose probe recovery, as L-glucose probe recovery remained unchanged. In a second set of experiments, the perfusion of 10 µM insulin did not increase the D/L ratio (1.40 ± 0.11) above that observed during 1.0 µM (1.41 ± 0.07) insulin perfusion. These data suggest that this method is sufficiently sensitive to detect differences in insulin-stimulated glucose uptake; thus the use of radioactive stereoisomers in conjunction with the microdialysis technique provides a novel and useful technique for determining tissue-specific glucose flux and insulin sensitivity.