We have sought evidence for blood-borne autoantigens from the apical regions of the intestinal epithelial cell that are associated with specific gastrointestinal diseases. While a wide range of brush border-specific proteins are antigenic in various disease states, significantly increased levels of autoantibody to villin, a 95-kDa protein microvillar actin-binding protein, were identified by western blot in colon cancer patients' sera. Examination of a population with a range of colonic diseases showed that anti-villin antibody is most prevalent in patients with colon cancer at significantly higher (P < 0.005) levels than normal controls. We conclude that cryptic antigens within the brush border, specifically including villin, incite an active autoimmune response. The pathological significance of these antibodies remains to be determined. These anti-villin antibodies also may provide a unique noninvasive approach for the detection of gastrointestinal pathology.