The L6 muscle cell line is proposed as an excellent cell culture system for studying glucose transport and its regulation by serum and insulin throughout myogenesis. The rate of hexose uptake decreased after cell fusion. Fetal calf serum (5 h) stimulated hexose uptake, and serum deprivation depressed it. Both effects were prevented by cycloheximide. Insulin stimulated the uptake of either 2-deoxy-D-glucose or 3-O-methyl-D-glucose provided that the basal rate was previously depressed by serum deprivation. The response to the hormone was significant only after the onset of cell fusion. Stimulation of transport was observed within the first 10 min after insulin administration and was insensitive to cycloheximide. Half-maximal stimulation of uptake was obtained with 5 X 10(-8) M insulin in 1% albumin solutions. Basal and insulin-stimulated uptake showed equal sensitivity to cytochalasin B. However, they differed in their sensitivity to extracellular Ca2+: basal transport was depressed, whereas insulin-stimulated transport was further enhanced in Ca2+-free media. Advantages of the cell culture system are discussed.