Seven subjects cycled to fatigue [75 +/- 5 (SE) min] at a work load corresponding to approximately 75% of their maximal oxygen uptake. Biopsies were taken from the quadriceps femoris muscle at rest and during exercise. Muscle glycogen decreased from a preexercise level of 445 +/- 33 mmol glucosyl units/kg dry wt to 50 +/- 14 at fatigue. The sum of the measured tricarboxylic acid cycle intermediates (TCAI = malate + citrate + fumarate + oxaloacetate) was 0.49 +/- 0.05 mmol/kg dry wt at rest, increased to 4.41 +/- 0.23 after 5 min of exercise, and then decreased continuously to 3.33 +/- 0.29 and to 2.83 +/- 0.27 mmol/kg dry wt after 40 min of exercise and at fatigue (P less than 0.05 vs. 5 min), respectively. The point of fatigue was characterized by an enhanced deamination of AMP (judged by increase in IMP) and reduced contents (vs. 5 min of exercise) of lactate, pyruvate, and alanine. In contrast, acetylcarnitine (reflects the availability of acetylunits) increased threefold at the onset of exercise and was maintained approximately at this level until fatigue. It is concluded that prolonged exercise to fatigue at moderate work loads results in glycogen depletion, energy deficiency (increased AMP deamination), reduced levels of three-carbon compounds and TCAI (compared with the initial phase of exercise) but in maintained levels of acetylunits. The present data indicate that carbohydrate depletion may impair aerobic energy production by reducing the level of TCAI.