Carbonic anhydrase (CA) inhibition is associated with a lower plasma lactate concentration ([La]_pl), but the mechanism for this association is not known. The effect of CA inhibition on muscle high-energy phosphates [ATP and phosphocreatine (PCr)], lactate ([La]_m), and glycogen was examined in seven men [28 ± 3 (SE) yr] during cycling exercise under control (Con) and acute CA inhibition with acetazolamide (Acz; 10 mg/kg body wt iv). Subjects performed 6-min step transitions in work rate from 0 W to a work rate corresponding to ~50% of the difference between the O₂ uptake at the ventilatory threshold and peak O₂ uptake. Muscle biopsies were taken from the vastus lateralis at rest, at 30 min postinfusion, at end exercise (EE), and at 5 and 30 min postexercise. Arterialized venous blood was sampled from a dorsal hand vein and analyzed for [La]_pl. ATP was unchanged from rest values; no difference between Con and Acz was observed. The fall in PCr from rest [72 ± 3 and 73 ± 3.6 (SE) mmol/kg dry wt for Con and Acz, respectively] to EE (51 ± 4 and 46 ± 5 mmol/kg dry wt for Con and Acz, respectively) was similar in Con and Acz. At EE, glycogen (mmol glucosyl units/kg dry wt) decreased to similar values in Con and Acz (307 ± 16 and 300 ± 19, respectively). At EE, no difference was observed in [La]_m between conditions (46 ± 6 and 43 ± 5 mmol/kg dry wt for Con and Acz, respectively). EE [La]_pl was higher during Con than during Acz (11.4 ± 1.0 vs. 8.2 ± 0.6 mmol/l). The similar [La]_m but lower [La]_pl suggests that the uptake of La by other tissues is enhanced after CA inhibition.