Fermentation in the large bowel has been postulated to play a protective role against colon cancer. Hydrogen and methane are end products of this fermentation process and are absorbed into the bloodstream and excreted via expired air in the breath. Breath levels of hydrogen and, to a lesser extent, methane correlate strongly with colonic fermentation and may serve as useful biomarkers for this process. In a preliminary study to assess the usefulness of these two markers in epidemiologic studies, we followed the hourly excretion of the two gases in expired alveolar air for 48 hr in 20 healthy subjects, using a Quintron gas chromatograph equipped with a solid-state detector specific for reducing gases. All subjects excreted hydrogen, but 71% did not excrete methane. Possible atmospheric contamination of the samples was corrected for on the basis of breath carbon dioxide levels. A clear circadian pattern of excretion was observed for breath hydrogen, with a decrease during the early morning followed by a progressive increase during the rest of the day. Methane excretion was constant throughout the day. This study shows that four samples collected at convenient times (0600, 1300, 1800, and 2200 hr) are optimal to characterize individuals by their breath excretions of hydrogen and methane during a 24-hr period.