Insulin-stimulated glucose uptake in skeletal muscle is mediated through the GLUT4 glucose transporter. Transgenic (TG) mice overexpressing human GLUT4 in skeletal muscle show an increased ability to handle a glucose load. Here, the participation of the overexpressed GLUT4 in the response to insulin was examined. In TG mouse muscle, the GLUT4 protein content was 10-fold higher in crude membrane (CM), sevenfold higher in internal membrane (IM), and 15-fold higher in a plasma membrane (PM)-rich fraction, relative to non-TG littermates. This suggested partial saturation of the normal sorting mechanisms. The distribution and abundance of the GLUT1 glucose transporter was not affected. Insulin injection (4.3 U/kg body wt) increased GLUT4 in the PM-rich fraction; the increase was threefold higher in TG than in non-TG mice. Insulin decreased the GLUT4 content of the IM in both animal groups and of a second, heavier intracellular membrane fraction only in TG mice. The net content of Na+-K+-pump subunits was 40-65% lower in CM from TG compared with non-TG littermates. In spite of this, insulin caused a three- to sixfold higher translocation of the alpha2 and beta1 subunits of the Na+-K+-pump in TG compared with non-TG animals. The results suggest that overexpression of GLUT4 confers to the muscle increased ability to translocate subunits of the Na+-K+-pump either as a direct consequence of the recruitment of glucose transporters or as an adaptation to the more demanding metabolic state.