The influence of metabolic substrates on the formation of volatile compounds by the colonic flora was measured in a fecal incubation system. The presence of carbohydrates (0, 25, and 50 mg/20 mL fecal suspension) led to a dose-related increase in the formation of alcohols and H2 and to a dose-related decrease in the formation of toxic mercaptans. This effect seemed to be independent of pH. The presence of albumin or fat (50 mg/20 mL fecal suspension) as substrates for the colonic flora gave rise to the formation of significantly higher amounts of methanethiol. Small amounts of pentane were found in the headspace after incubation with oil. These data show that the formation of volatile metabolites by the colonic flora is greatly influenced by the available substrates.