Insulin-stimulated glucose transport in skeletal muscle is regarded as a key determinant of insulin sensitivity, yet isolation of this step for quantification in human studies is a methodological challenge. One notable approach is physiological modeling of dynamic positron emission tomography (PET) imaging using 2-[18-fluoro]2-deoxyglucose ([(18)F]FDG); however, this has a potential limitation in that deoxyglucose undergoes phosphorylation subsequent to transport, complicating separate estimations of these steps. In the current study we explored the use of dynamic PET imaging of [(11)C]3-O-methylglucose ([(11)C]3-OMG), a glucose analog that is limited to bidirectional glucose transport. Seventeen lean healthy volunteers with normal insulin sensitivity participated; eight had imaging during basal conditions, and nine had imaging during euglycemic insulin infusion at 30 mU/min.m(2). Dynamic PET imaging of calf muscles was conducted for 90 min after the injection of [(11)C]3-OMG. Spectral analysis of tissue activity indicated that a model configuration of two reversible compartments gave the strongest statistical fit to the kinetic pattern. Accordingly, and consistent with the structure of a model previously used for [(18)F]FDG, a two-compartment model was applied. Consistent with prior [(18)F]FDG findings, insulin was found to have minimal effect on the rate constant for movement of [(11)C]3-OMG from plasma to tissue interstitium. However, during insulin infusion, a robust and highly significant increase was observed in the kinetics of inward glucose transport; this and the estimated tissue distribution volume for [(11)C]3-OMG increased 6-fold compared with basal conditions. We conclude that dynamic PET imaging of [(11)C]3-OMG offers a novel quantitative approach that is both chemically specific and tissue specific for in vivo assessment of glucose transport in human skeletal muscle.