Three dietary fiber sources (corn fiber, oat bran, wheat bran) were analyzed for chemical composition and potential fermentation by human colonic bacteria in vitro. Total dietary fiber (TDF) concentration of substrates was 64.3, 11.1 and 50.4 g/100 g dry matter for corn fiber, oat bran and wheat bran, respectively. Original material (ORIG), TDF fractions and simulated (SIM) cell wall fractions (produced by combining cellulose, hemicelluloses and pectic substances in proportions they represented in the cell wall) from each substrate were fermented in vitro for 6, 12, 18, 24 or 48 h using inoculum prepared from freshly voided feces from each of three human volunteers. Substrate dry matter remaining after 48 h of fermentation was 87.8, 39.8 and 73.5% for TDF fractions of corn fiber, oat bran and wheat bran, respectively. Disappearance of ORIG fractions was considerably greater than that of TDF due to fermentation of nonfibrous material. Disruption of cell wall structure during isolation of polysaccharide fractions allowed for dramatically increased fermentability of SIM relative to TDF. Averaged across all treatments, production of the short-chain fatty acids, acetate, propionate and butyrate, occurred in the molar ratio 63:21:16; however, profiles of short-chain fatty acids produced were influenced by both treatment and inoculum source. Extent of substrate fermentation varied among inoculum donors, implying that colonic microbial activities differ among individuals. Potential colonic fermentability of fiber sources was influenced by substrate, method of fiber preparation and inoculum source.