In cattle, Escherichia coli O157 generally persists in the large intestine more often than in the rumen. In contrast to the rumen, the large intestine is lined by an epithelial membrane that secretes mucus. We hypothesize that substrates contained in intestinal mucus may constitute a source of energy that is preferentially used by E. coli O157. Therefore, our objective was to test the effects of mucin and its carbohydrate constituents on in vitro growth of E. coli O157 in ruminal or fecal microbial fermentations. Ruminal contents and feces were collected from a ruminally cannulated donor steer fed a corn grain-based finishing diet. Ruminal contents were strained through 2 layers of cheesecloth and incubated at 39 degrees C for 1 h; the floating hay mat was removed with a vacuum suction; and the remaining material was utilized as rumen microbial inoculum. Feces were suspended in physiologic saline to increase fluidity, blended, and strained through 2 layers of cheesecloth. The resulting fluid was utilized as fecal microbial inoculum. Fermentations (50 mL) were performed in serum bottles with a 2:1 mineral buffer to microbial inoculum ratio. Substrates (mucin, fucose, galactose, mannose, gluconic acid, galacturonic acid, glucuronic acid, galactosamine, and glucosamine) were added at 10 mg/mL. A mixture of 5 strains of nalidixic acid-resistant (Nal(R)) E. coli O157 strains was added to each fermentation and concentrations were determined after 0, 6, 12, and 24 h of incubation. In ruminal fermentations, fucose, mannose, glucuronic acid, galacturonic acid, glucosamine, galactosamine, and mucin had no effect on Nal(R) E. coli O157 concentration compared with the control (no substrate added) fermentation. At 24 h of fermentation, the mean concentration of Nal(R) E. coli O157 in fermentations with galactose was less than the control. However, including gluconic acid as substrate increased Nal(R) E. coli O157 concentration at 24 h. In fecal fermentations, mannose, galactose, gluconic acid, glucuronic acid, galacturonic acid, glucosamine, and mucin increased Nal(R) E. coli O157 growth compared with control at 24 h, whereas galactosamine and fucose did not. Gluconic acid was the most stimulatory substrate, increasing Nal(R) E. coli O157 by more than 1.0 log in ruminal fermentations and 2.0 log in fecal fermentations. In summary, availability of mucous constituents, particularly gluconic acid, may explain the greater prevalence of E. coli O157 in the large intestine compared with the rumen of the digestive tract.