Acetazolamide (AZ) is a medication commonly used to prevent acute mountain sickness (AMS) during rapid ascent to high altitude. However, it is unclear whether AZ use impairs exercise performance; previous literature regarding this topic is equivocal. The purpose of this study was to evaluate the impact of AZ on time-trial (TT) performance during a 30-h exposure to hypobaric hypoxia equivalent to 3,500-m altitude. Ten men [sea-level peak oxygen consumption (VO2peak): 50.8 +/- 6.5 mL.kg(-1).min(-1); body fat %: 20.6 +/- 5.2%] completed 2 30-h exposures at 3,500 m. In a crossover study design, subjects were given 500 mg/day of either AZ or a placebo. Exercise testing was completed 2 h and 24 h after ascent and consisted of 15-min steadystate treadmill walking at 40%- 45% sea-level VO(2)peak, followed by a 2-mile self-paced treadmill TT. AMS was assessed after similar to 12 h and 22 h at 3,500 m. The incidence of AMS decreased from 40% with placebo to 0% with AZ. Oxygen saturation was higher (P < 0.05) in AZ versus placebo trials at the end of the TT after 2 h (85 +/- 3% vs. 79 +/- 3%) and 24 h (86 +/- 3% vs. 81 +/- 4%). There was no difference in time to complete 2 miles between AZ and PL after 2 h (20.7 +/- 3.2 vs. 22.7 +/- 5.0 min, P > 0.05) or 24 h (21.5 +/- 3.4 vs. 21.1 +/- 2.9 min, P > 0.05) of exposure to altitude. Our results suggest that AZ (500 mg/day) does not negatively impact endurance exercise performance at 3,500 m. NEW & NOTEWORTHY To our knowledge, this is the first study to examine the impact of acetazolamide (500 mg/day) versus placebo on self-paced, peak-effort exercise performance using a short-duration exercise test in a hypobaric hypoxic environment with a repeatedmeasures design. In the present study, acetazolamide did not impact exercise performance after 2-h or 24-h exposure to 3,500-m simulated altitude.