The content of glucose 1,6-bisphosphate (G-1,6-P2), an in vitro activator of phosphofructokinase (a rate-limiting enzyme for glycolysis), and the glycolytic rate in skeletal muscle during isometric contraction have been determined. Subjects contracted the knee extensor muscles at two-thirds maximal voluntary force to fatigue. Biopsies from the quadriceps femoris muscle were obtained before and immediately after contraction. G-1,6-P2 increased in all subjects from a mean of 101 +/- 15 (SE) mumol/kg dry wt at rest to 128 +/- 24 at fatigue (P less than 0.05). Muscle glucose did not change significantly, whereas hexosemonophosphates were significantly increased after contraction. The glycogenolytic and glycolytic rate averaged 70.0 +/- 13.8 and 47.3 +/- 6.7 mmol.kg dry wt-1.min-1, respectively, and the glycolytic rate was positively correlated with the accumulation rates of fructose 6-phosphate (F-6-P) (r = 0.95, P less than 0.01) and G-6-P (r = 0.96, P less than 0.01). Phosphocreatine and ATP decreased by 87 and 17%, respectively, whereas ADP increased by 31% after contraction. These data demonstrate that intense, short-term isometric contraction results in an elevation of the muscle content of G-1,6-P2. The increase in G-1,6-P2 could not be accounted for by the side reactions of phosphoglucomutase or phosphofructokinase. It remains to be determined whether the observed increase in G-1,6-P2 is sufficient to account for the high glycolytic rate during intense exercise. The lack of increase in muscle glucose while G-6-P increased (which will inhibit hexokinase) suggests that the debranching enzyme complex was not active during contraction.