The effect of bicycle exercise (75% of maximal oxygen uptake) on glucose uptake by the inferior limb (LGU) and glycolysis in human skeletal muscle has been investigated. Biopsies were obtained from the quadriceps femoris muscle before exercise, after 5 and 40 min of exercise, and at fatigue [74.9 +/- 4.7 (SE) min]. LGU was 0.05 +/- 0.02 mmol/min at rest, increased approximately sevenfold after 5 min of exercise, and continued to increase linearly during the first 40 min of exercise. Thereafter LGU stabilized at approximately 1.4 mmol/min until fatigue. Intracellular glucose was low at rest but increased sixfold after 5 min of exercise (P less than 0.01 vs. rest); thereafter, intracellular glucose decreased and was not significantly different from the value at rest after 40 min or at fatigue (P greater than 0.05). D-Glucose 6-phosphate (G-6-P) and alpha-D-glucose 1,6-bisphosphate (G-1,6-P2) (inhibitors of hexokinase) increased significantly after 5 min of exercise (approximately 300% G-6-P; approximately 25% G-1,6-P2) and then decreased continuously. The muscle glycolytic rate (glycogenolysis + glucose uptake) averaged 7.7 mmol.kg dry wt-1.min-1 during the first 40 min of exercise and 3.7 mmol.kg dry wt-1.min-1 during the last 35 min of exercise. The contribution of extracellular glucose to muscle glycolysis was estimated to be only 5 and 19% during the initial and latter phases of exercise, respectively. It is concluded that, during the initial phase of exercise, glucose utilization is limited by phosphorylation, probably due to G-6-P-dependent inhibition of hexokinase.(ABSTRACT TRUNCATED AT 250 WORDS)