This study examined the relationship between preexercise muscle glycogen content and glycogen utilization in two physiological pools, pro- (PG) and macroglycogen (MG). Male subjects ( n = 6) completed an exercise and dietary protocol before the experiment that resulted in one leg with high glycogen (HL) and one with low glycogen (LL). Preexercise PG levels were 312 ± 29 and 208 ± 31 glucosyl units/kg dry wt (dw) ( P ≤ 0.05) in the HL and LL, respectively, and the corresponding values for MG were 125 ± 37 and 89 ± 43 mmol glucosyl units/kg dw ( P ≤ 0.05). Subjects then performed two 90-s exercise bouts at 130% maximal oxygen uptake separated by a 10-min rest period. Biopsies were obtained at rest and after each exercise bout. Preexercise glycogen concentration was correlated to net glycogenolysis for both PG and MG for bout 1 and bouts 1 and 2 ( r ≤ 0.60). In bout 1, there was no difference in the rate of PG or MG catabolism between HL and LL despite a 26% increase ( P ≤ 0.05) in glycogen phosphorylase transformation ( phos a %) in the HL. In the second bout, more PG was catabolized in the HL vs. LL (38 ± 9 vs. 9 ± 6 mmol glucosyl units · kg dw−1 · min−1) ( P ≤ 0.05) with no difference between legs in phos a %. phos a% was increased in HL vs. LL but does not necessarily increase glycogenolysis in either PG or MG. Despite both legs performing the same exercise and having identical metabolic demands, the HL catabolized 2.3 ( P ≤ 0.05) times more PG and 1.5 ( P ≤ 0.05) times more MG vs. LL in bouts 1 and 2, indicating that preexercise glycogen concentration is a regulator of glycogenolysis.