Skeletal muscle glucose transport and metabolism were studied in a line of transgenic mice overexpressing the human Glut4 facilitative glucose transporter. Skeletal muscle Glut4 protein levels were increased 2-4-fold in transgenic animals relative to their nontransgenic litter mates. Glut4 overexpression increased total transport activity (measured with 1 mM 2- deoxy-D-glucose) in the isolated extensor digitorum brevis muscle in the presence of insulin; this increase was due to 1) an increase in basal glucose transport (0.8 ± 0.1 versus 0.5 ± 0.1 μmol · ml-1 · 20 min-1 in transgenic and control mice, respectively) and 2) an increase in insulin- stimulated transport (1.5 ± 0.1 versus 0.8 ± 0.1 μmol · ml-1 · 20 min-1 above basal transport in transgenic and control mice, respectively). Glut4 overexpression also increased glucose transport stimulated by muscle contractions. In addition, glycolysis and glucose incorporation into glycogen were enhanced in muscle isolated from transgenic mice compared to controls. These data demonstrate that Glut4 overexpression in skeletal muscle increases insulin- and contraction-stimulated glucose transport activity and glucose metabolism. These findings are consistent with the role of Glut4 as the primary mediator of transport stimulated by insulin or contractions.