BACKGROUND: Known risk factors or epidemiological associations for necrotizing enterocolitis (NEC) are not usually incorporated in the development of animal models of the disease. We attempted to develop a rat model that was reliable and incorporated several risk factors for the development of NEC. Of primary interest was gut colonization, feeding and poor intestinal motility. We used adult rats for technical reasons and therefore the risk factor of prematurity remained unaccounted. METHODS: Holtzman rats (220-280g) were anesthetized and under aseptic conditions a midline laparotomy was performed and the ileum was exposed. Dysmotility was achieved by forming an intestinal loop with silk ligatures and a cocktail of casein-dominant infant formula and E. coli, obtained from an infant with NEC (1 million CFU/ml), was administered into the lumen by transmural injection. Control animals received either the formula alone or E. coli without substrate. The E. coli isolate rapidly fermented carbohydrates to organic acids and was resistant to low doses of ampicillin. After closing the abdominal cavity the animals were allowed to recover. After 24 h rats were reanesthetized, the ileum isolated and segments obtained for cytokine and inducible nitric oxide synthase (iNOS) gene expression (RTR-PCR), routine histology and immunohistochemistry for iNOS and myeloperoxidase activity for estimation of granulocyte infiltration. RESULTS: Rats treated with a combination of fast fermenting E. Coli and substrate(formula) developed NEC which ranged from villus tip injury to hemorrhagic necrosis and perforations. In some animals pneumatosis intestinalis was detected. In control animals the intestine was not injured. Granulocyte infiltration was largely serosal and submucosal and associated with iNOS expression. Gene expression for iNOS, IL-10 and TNFα were enhanced in NEC rats. CONCLUSION: This model demonstrates that the rapid conversion of substrate to acids by otherwise normal flora in a confined gut segment can lead to NEC. We anticipate that the continued delivery of substrate, mimicking rapid advances in feeding, would lead to further intestinal injury.