To examine the effects of alterations in preexercise muscle glycogen availability on glycogenolysis and glucose uptake during exercise, 12 active but untrained men [22.8 +/- 1.6 (SE) yr, 71.7 +/- 2.0 kg, peak pulmonary oxygen uptake 3.85 +/- 0.16 l/min] were studied during 40 min of cycle ergometer exercise at 65-70% peak pulmonary oxygen uptake on two separate occasions, at least 1 wk apart. Preexercise muscle glycogen concentrations were manipulated by having the subjects perform glycogen-lowering exercise either 24 or 48 h before a trial, in combination with either high or low dietary carbohydrate intake. In series 1 (n = 7), increasing muscle glycogen from 90.3 +/- 6.0 to 124.7 +/- 10.8 mmol/kg wet wt increased muscle glycogenolysis during exercise (62.7 +/- 7.9 vs. 49.1 +/- 6.6 mmol/kg; P < 0.05). Similarly, in series 2 (n = 5) when muscle glycogen was reduced from 96.2 +/- 6.6 to 53.7 +/- 6.0 mmol/kg, glycogen utilization during exercise was reduced from 51.8 +/- 4.6 to 28.3 +/- 3.8 mmol/kg (P < 0.05). The altered muscle glycogen utilization was associated with alterations in carbohydrate oxidation during exercise, without effect on tracer ([3H]glucose)-determined glucose uptake. These results indicate that preexercise muscle glycogen availability influences muscle glycogenolysis, but not glucose uptake, during exercise.