This study examined the effects of caffeine (Caf) ingestion on muscle glycogen use and the regulation of muscle glycogen phosphorylase (Phos) activity during intense aerobic exercise. In two separate trials, 12 untrained males ingested either placebo (Pl) or Caf (9 mg/kg body wt) 1 h before cycling at 80% maximum O2 consumption (V˙o 2 max) for 15 min. Muscle biopsies were obtained from the vastus lateralis at 0, 3, and 15 min of exercise. In this study, glycogen “sparing” was defined as a 10% or greater reduction in muscle glycogen use during exercise after Caf ingestion compared with Pl. Muscle glycogen use decreased by 28% (Pl 255 ± 38 vs. Caf 184 ± 24 mmol/kg dry muscle) after Caf in six subjects [glycogen sparers (Sp)] but was unaffected by Caf in six other subjects [nonsparers (NSp), Pl 210 ± 35 vs. Caf 214 ± 37 mmol/kg dry muscle]. In both groups, Caf significantly increased resting free fatty acid concentration, significantly increased epinephrine concentration by twofold during exercise, and increased the Phos a mole fraction at 3 min of exercise compared with Pl, although not significantly. Caf improved the energy status of the muscle during exercise in the Sp group: muscle phosphocreatine (PCr) degradation was significantly reduced (Pl 47.9 ± 3.6 vs. Caf 40.4 ± 6.7 mmol/kg dry muscle at 3 min) and the accumulations of free ADP and free AMP (Pl 6.8 ± 1.3 vs. Caf 3.1 ± 1.4 μmol/kg dry muscle at 3 min; Pl 8.7 ± 0.8 vs. Caf 4.7 ± 1.1 μmol/kg dry muscle at 15 min) were significantly reduced. Caf had no effect on these measurements in the NSp group. It is concluded that the Caf-induced decrease in flux through Phos (glycogen-sparing effect) is mediated via an improved energy status of the muscle in the early stages of intense aerobic exercise. This may be related to an increased availability of fat and/or ability of mitochondria to oxidize fat during exercise preceded by Caf ingestion. It is presently unknown why the glycogen-sparing effect of Caf does not occur in all untrained individuals during intense aerobic exercise.