Human muscle cells were grown in culture from satellite cells present in muscle biopsies and fusion-competent clones were identified. Hexose uptake was studied in fused myotubes of human muscle cells in culture and compared with hexose uptake in myotubes of the rat L6 and mouse C2C12 muscle cell lines. Uptake of 2-deoxyglucose was saturable and showed an apparent Km of about 1.5 mM in myotubes of all three cell types. The Vmax of uptake was about 6000 pmol/(min.mg protein) in human cells, 4000 pmol/(min.mg protein) in mouse C2C12 muscle cells, and 500 pmol/(min.mg protein) in L6 cells. Hexose uptake was inhibited approximately 90% by cytochalasin B in human, rat, and mouse muscle cell cultures. Insulin stimulated 2-deoxyglucose uptake in all three cultures. The hormone also stimulated transport of 3-O-methylglucose. The sensitivity to insulin was higher in human and C2C12 mouse myotubes (half-maximal stimulation observed at 3.5 X 10(-9) M) than in rat L6 myotubes (half-maximal stimulation observed at 2.5 X 10(-8) M). However, insulin (10(-6) M) stimulated hexose uptake to a larger extent (2.37-fold) in L6 than in either human (1.58-fold) or mouse (1.39-fold) myotubes. It is concluded that human muscle cells grown in culture display carrier-mediated glucose uptake, with qualitatively similar characteristics to those of other muscle cells, and that insulin stimulates hexose uptake in human cells. These cultures will be instrumental in the study of human insulin resistance and in investigations on the mechanism of action of antidiabetic drugs.