Germfree rats monocontaminated with the anaerobic microorganisms Clostridium difficile or another Clostridium species (strain G 62) produce auto-antibodies to colon antigen. The antigen can be extracted with phenol water from the feces of germfree rats. Antibodies, demonstrable by means of passive hemagglutination of antigen sensitized sheep erythrocytes appear after monocontamination for 35 days or longer. The indirect immunofluorescence techniques, applied to sections of germfree rat colon, gave positive mucosal staining. The staining was similar to that obtained with sera from patients with ulcerative colitis or from rats immunized with rabbit colon. No antibodies were found in the sera of germfree rats, germfree rats monocontaminated with various other bacteria, conventional rats of germfree origin, or conventional Sprague-Dawley rats. Although the anti-colon antibodies of the Clostridium infected rats reacted with the same feces extract as the antibodies of ulcerative colitis patients or of rabbit colon immunized rats, their specificity was different. While the latter cross-react with polysaccharide from E. coli O14, those from the Clostridium-infected exgermfree rats did not. Rats monocontaminated with Cl. difficile also developed antibodies to this organism, but no cross-reaction between Cl. difficile antigen and colon antigen could be demonstrated. This speaks against breakage of tolerance by cross-reacting bacterial antigen as the cause of autoimmunity in these rats. Other possible mechanisms for autoantibody production in this model are immunogenic alteration of gastrointestinal mucins by bacterial degradation, adjuvant effects of bacterial products, or both.