Previous observations have purported to demonstrate circulating antibodies which bind to colon epithelial cells. However, the significance and reproducibility of such observations has been difficult and the data often phenomenological. To further our understanding of such autoreactivity, we studied sera and purified serum immunoglobulins from patients with ulcerative colitis, Crohn's colitis and other inflammatory diseases, as well as normal volunteers using as a target, well-defined epithelial cell preparations from normal and diseased colon and small bowel including crude suspensions of homogenized cells, purified and characterized brush border membranes, basolateral membranes and a DEAE cellulose column purified protein fraction. Homogenates of normal liver, lung, kidney, thymus, pancreas, stomach and small and large intestine, obtained at surgery, were also included. The purified preparations were characterized by enzyme activity and were electrophoresed on SDS-polyacrylamide gels for immunoblotting. Additional studies were carried out comparing these findings with those of a previously published and described 'positive' colon target preparation and polyclonal antibody. There was no convincing demonstration of circulating autoantibodies in patients with ulcerative colitis. Our data, using well-defined and characterized tissue preparations, raises doubts regarding the presumptive demonstration of autoantibodies in ulcerative colitis.