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Colonic bacterial production of short-chain fatty acids (SCFA) plays an important role in the salvage of unabsorbed carbohydrate and in colonic absorption of electrolytes and water. The objective of this study was to determine whether patients with diarrhea-predominant irritable bowel syndrome (DP-IBS) have a different pattern and rate of fermentation of carbohydrate and fiber to SCFA compared with controls. Fecal homogenates from 10 patients with DP-IBS and 10 age-matched controls were studied. SCFA were measured by gas chromatography in baseline fecal samples and in fecal homogenates in an in vitro anaerobic fermentation system after incubation with no additional substrate, lactulose, potato starch, citrus pectin, and hemicellulose over a 24-hour period. Net SCFA production rates were calculated for the first 6 h of the incubation period. Patients with DP-IBS had a consistently different pattern of less total SCFA, a lower percentage of acetate (p < 0.05), and a higher proportion of n-butyrate (p < 0.05) than controls. In stool homogenates from both controls and DP-IBS patients, lactulose fermentation resulted in the highest rate of SCFA production followed by pectin, starch, and hemicellulose. However, at all time points, the fecal homogenates from controls generated a higher concentration of total SCFA, acetate, and propionate with all substrates tested. SCFA production rates were higher in controls incubated with lactulose, starch, and hemicellulose. The fecal SCFA profile of patients with DP-IBS is characterized by lower concentrations of total SCFA, acetate, and propionate and a higher concentration and percentage of n-butyrate. Fecal flora from these patients produced less SCFA in an in vitro fermentation system in response to incubations with various carbohydrates and fibers. Differences in SCFA production by colonic bacterial flora in patients with DP-IBS may be related to the development of gastrointestinal symptoms.