Hydrogen produced by colonic bacteria and excreted in breath is a useful index of carbohydrate malabsorption. Since colonic contents are often acidic in individuals with carbohydrate malabsorption and in normal newborns, we determined the effect of colonic acidification on H2 production. Acidification of colonic contents by dietary means significantly reduced excess breath H2 excretion from 55.4 +/- 11.1 (SEM) to 12.2 +/- 3.1 ml/4 h (P less than 0.05) after administration of 0.3 g/kg of the nonabsorbable sugar lactulose to five normal adult subjects. Similarly, the breath H2 response to lactose was reduced or eliminated in two proven lactose malabsorbers after acidification. The correlation between pH and H2 production from carbohydrate was further investigated in adults and neonates, using an in vitro fecal incubation system. Glucose disappearance and H2 production were pH dependent and highly correlated (r = 0.94) in the pH range 5.5-7.6. Maximal production of H2 from glucose by fecal incubates occurred at pH 7.0-7.45. Inhibition of H2 production from carbohydrate occurred at acid pH. H2 per hour from glucose at pH 6.2 and 5.5 averaged 60.2% and 24.2%, respectively, of that produced at neutral pH. Rapid reversal of pH-induced inhibition by neutralization indicated a metabolic, rather than a bactericidal process. The observations indicate that the breath H2 response to malabsorbed carbohydrate is affected by colonic pH. It appears that the efficiency of bacterial carbohydrate metabolism in the colon is pH dependent.