(1) Myoblasts in culture (L6 cell line) were used as an in vitro model system, to study the kinetic and pharmacological properties of hexose transport in skeletal muscle tissue. (2) Uptake of 2-deoxy-D-[3H]glucose into L6 cells grown in monolayer culture was judged rate limiting since: (2) The time course of sugar uptake extrapolated to zero, (b) a parallel inhibition of hexose uptake and phosphorylation was caused by cytochalasin B, and (c) very little backflow of the hexose was detected. (3) Uptake of 2-deoxy-D-[3H]glucose by cells in monolayers was linear for at least 20 min and it was stimulated by countertransport. The Kt value was 0.83 mM. Cytochalasin B inhibited uptake non-competitively, and half maximal inhibition was achieved at 0.3 microM. Cytochalasin E (up to 5 microM) did not affect 2-deoxy-D-[3H]glucose uptake. (4) L6 myoblasts, detached by trypsinization, retained the hexose transport activity. Kt in detached cells was 0.96 mM. V was 3.2 nmol/min per mg protein, and half maximal inhibition was observed with 0.25 microM cytochalasin B. (5) [3H]Cytochalasin B binding to detached cells showed saturable and non-saturable components. The former could be further separated into cytochalasin E-sensitive binding (probably associated to cytoskeletal proteins) and cytochalasin E-insensitive binding, a fraction of which was inhibited by D-glucose. The D-glucose sensitive sites amount to 16.3 pmol/mg protein, and showed a Kd of 0.49 microM, which is in close agreement with the Ki of cytochalasin B inhibition of hexose uptake. These sites probably are equivalent to the hexose carrier molecules, and are present at a density of 6.8 . 10(6) sites/cell.