Gastrointestinal dysfunction is a common secondary complication of insulin-dependent diabetes mellitus, yet its etiology is unclear. Enteric microbial overgrowth may play a role. To quantitate the changes in mucosal-adherent enteric microbial populations in untreated diabetes mellitus and to assess the impact of two forms of insulin replacement therapy upon enteric microbial populations, age-matched male Lewis rats were rendered diabetic by the administration of intravenous streptozotocin (55 mg/kg). After diabetes was confirmed (blood glucose level greater than 250 mg/dL), rats were divided into three groups: no treatment (no insulin), treatment with daily insulin to maintain normoglycemia (3 to 7 units of protamine zinc insulin subcutaneously), or transplantation with a vascularized heterotopic duct-ligated pancreatic isograft. After 1 month, rats were killed, and segments of the proximal, middle, and distal small bowel were obtained. Mucosal samples were rinsed in phosphate-buffered saline to remove nonadherent bacteria prior to aerobic and anaerobic culturing. Microbial recovery was expressed as the log10 colony-forming unit/mg tissue wet weight. Untreated diabetes resulted in an overgrowth of mucosal-associated small bowel aerobic and anaerobic microbial populations compared with populations in normal nondiabetic age-matched control rats. Insulin treatment and pancreatic transplantation prevented microbial overgrowth in the diabetic small intestine. Pancreatic transplantation resulted in strict normoglycemia equivalent to that in nondiabetic control rats, whereas insulin treatment resulted in slightly higher blood glucose levels at sacrifice and wide fluctuations in blood glucose levels compared with nondiabetic control rats. These data suggest that sustained normalization of glucose levels is not required to prevent microbial overgrowth in diabetic rats.