Background and Aims: Clostridium difficile-associated diarrhea is a major medical problem throughout the world. A widely-practiced technique in the evaluation of chronic diarrhea is perform colonoscopy/sigmoidoscopy and to aspirate stool for enzyme immunoassay (EIA) to test for C. difficile. At our institution, we observed an unexpectedly high proportion of positive C. difficile assays on stool obtained during colonoscopy, often following a negative stool test. Our goals were to evaluate for false positive C. difficile EIA tests in colonoscopy aspirates and to determine possible etiologies of false-positivity. Methods: We obtained colonoscopy aspirates from 40 consecutive patients who presented for routine colorectal cancer screening. These patients had no complaints of diarrhea prior to bowel preparation. Residual stool and liquid were aspirated into a stool trap during colonoscopy from a single physician and nurse. Stool samples were then sent to a blinded microbiologist for routine C. difficile EIA testing (CDIFF TOX A/B II, TechLab/Wampole, Blacksburg, VA). Positive EIA tests were followed-up with tissue culture cytotoxin assay for confirmation. Finally, commonly used items in colonoscopy, including simethicone, lubricant, and bowel preparation (Polyethylene Glycol 3350 - TriLyte™ with flavor packs, Schwarz Pharma, Inc. Milwaukee, WI) mixed in sterile water, were also sent individually for routine C. difficile EIA testing. Results: Stool aspirates were processed successfully in 38 cases. EIA assay for toxins A or B was positive in 27 of 38 cases (67%). None were positive by either confirmatory tissue culture cytotoxin assays or culture for C. difficile. Further, sampling of Polyethylene Glycol 3350 bowel prep mixed with sterile water in normal dilution (but no stool), as well as simethicone alone, tested positive for C. difficile by EIA. Conclusions: We found a substantial proportion of false positive C. difficile assays on aspirates obtained during colonoscopy. A potential explanation may be that PEG-based bowel preparations and/or simethicone interact with the reagents in an EIA well-based assay, which lead to false-positive findings. We feel that this is relevant and vital information for all practicing gastroenterologists. Further study is being undertaken to determine whether this phenomenon is applicable to all combinations of bowel preparation and other commercially-available C. difficile assays.