In muscle, insulin enhances influx of glucose and its conversion to glucose 6-phosphate (G6P) by hexokinase (HK). While effects of insulin on glucose transport have been demonstrated, its effect on the activity of HK of cells has not. In L6 myotubes treated for 24 h with insulin there was increased expression of the HK isoform, HKII, and increased glucose phosphorylation without a concomitant increase in glucose transport, indirectly suggesting that phosphorylation of glucose was a target of insulin action [Osawa, Printz, Whitesell and Granner (1995) Diabetes 44, 1426-1432]. In the present work the same treatment led to a 2-fold rise in G6P, suggesting that transport and/or HK were important targets of insulin action. We used a method to identify the site of rate control involving the specificity of phosphorylation towards 2-deoxy-[1-14C]glucose and D-[2-3H]glucose. Glucose transport does not greatly discriminate between these two tracers while HK shows increased specificity for glucose. Specificity of the glucose phosphorylation of the cells increased with addition of insulin and when extracellular glucose was raised. Specificity was reduced at low glucose concentrations or when the inhibitor of transport, cytochalasin B, was added. We conclude that transport and HK share nearly equal control over glucose phosphorylation in these cells. A computer program was used to test models for compatibility with the different types of experiments. The predicted intracellular glucose and transport rates associated with phosphorylation activity were lower than their measured values for the whole cell. In the most likely model, 15+/-4% of the glucose transporters serve a proportionate volume of the cytoplasm. Insulin activation of glucose phosphorylation might then result from stimulation of these transporters together with HK recruitment or relief from inhibition by G6P.