Skeletal muscle glucose transport was examined in transgenic mice overexpressing the glucose transporter GLUT1 using both the isolated incubated-muscle preparation and the hind-limb perfusion technique. In the absence of insulin, 2-deoxy-d-glucose uptake was increased ∼ 3–8-fold in isolated fast-twitch muscles of GLUT1 transgenic mice compared with non-transgenic siblings. Similarly, basal glucose transport activity was increased ∼ 4–14-fold in perfused fast-twitch muscles of transgenic mice. In non-transgenic mice insulin accelerated glucose transport activity ∼ 2–3-fold in isolated muscles and to a much greater extent (∼ 7–20-fold) in perfused hind-limb preparations. The observed effect of insulin on glucose transport in transgenic muscle was similarly dependent upon the technique used for measurement, as insulin had no effect on isolated fast-twitch muscle from transgenic mice, but significantly enhanced glucose transport in perfused fast-twitch muscle from transgenic mice to ∼ 50–75% of the magnitude of the increase observed in non-transgenic mice. Cell-surface glucose transporter content was assessed via 2-N-4-(l-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(d -mannos-4-yloxy)-2-propylamine photolabelling methodology in both isolated and perfused extensor digitorum longus (EDL). Cell-surface GLUT1 was enhanced by as much as 70-fold in both isolated and perfused EDL of transgenic mice. Insulin did not alter cell-surface GLUT1 in either transgenic or non-transgenic mice. Basal levels of cell-surface GLUT4, measured in either isolated or perfused EDL, were similar in transgenic and non-transgenic mice. Interestingly, insulin enhanced cell-surface GLUT4 ∼ 2-fold in isolated EDL and ∼ 6-fold in perfused EDL of both transgenic and non-transgenic mice. In summary, these results reveal differences between isolated muscle and perfused hind-limb techniques, with the latter method showing a more robust responsiveness to insulin. Furthermore, the results demonstrate that muscle overexpressing GLUT1 has normal insulin-induced GLUT4 translocation and the ability to augment glucose-transport activity above the elevated basal rates.