Hydrogen and methane in human breath derive entirely from bacterial fermentation in the intestinal lumen. The sources of substrates utilized for these reactions have not been completely determined. Basal excretion of both gases occurs in the fasted state, while malabsorbed carbohydrate commonly results in increased hydrogen but not methane production. Using an in vitro fecal incubation system, we have studied hydrogen and methane production from glycoproteins of endogenous as well as dietary origin. All glycoproteins tested yielded hydrogen when incubated with fecal homogenates. Mean hydrogen production from substrates containing less than 3% sugar (human serum albumin, bovine serum albumin, and alpha-casein) averaged 2.2 +/- 0.9% of hydrogen production from equivalent amounts of glucose, while carbohydrate-rich mucin yielded 46.0 +/- 6.7% of hydrogen production from glucose. Glycoproteins of intermediate carbohydrate content, including transferrin and egg white, yielded intermediate values. Methane production from glycoproteins was optimal from carbohydrate-poor protein substrates in fecal homogenates which accumulated hydrogen and became rapidly acidic when incubated with pure carbohydrate. In contrast, methane production was comparable for essentially sugar-free proteins, glycoproteins, and glucose when hydrogen did not accumulate and neutral pH was maintained. We conclude that glycoproteins are substrates for hydrogen and methane production by colonic bacteria from healthy adults. In individuals with bacterial overgrowth syndromes and in protein-losing enteropathy, bacterial catabolism of endogenous glycoproteins may cause increased basal hydrogen and methane excretion. These findings also raise the possibility that measurement of hydrogen or methane after oral administration of dietary glycoproteins may be useful in detection of protein malabsorption.