Clostridium difficile infection is associated with substantial morbidity and mortality, increased duration of hospitalization, and a marked economic impact. Several case reports and case series have described C. difficile infection in excluded bowels or immediately after reversal of defunctioning ileostomy. The aim of this prospective study is to detect whether the excluded colon is associated with a higher rate of C. difficile colonization than the normal population, which may increase the risk of C. difficile infection. Patients with defunctioning loop ileostomy, undergoing closure of ileostomy to restore bowel continuity, were prospectively recruited. Two stool samples were collected from the ileostomy effluent before closure of ileostomy and two after the procedure including the first bowel movement. All samples were cultured for C. difficile and analyzed for toxins A and B by a Premier EIA test. Demographic data and possible confounding factors were observed and recorded. Twenty-fine adult patients were recruited to this study; five patients were subsequently excluded. Two patients had positive stool cultures for C. difficile in the postoperative samples and another patient developed clinical pseudomembranous colitis with positive toxin. This indicates a possible colonization rate of 3 to 38 per cent (95% confidence interval). Four observed cases out of the 20 subjects taking part in this study would confidently conclude that C. difficile colonization in the excluded colon is 6 to 44 per cent, i.e., higher than the incidence in the healthy adult population, which is 3 per cent. However, the findings of this study prompt larger and well-powered studies to confirm these findings.