Anti-alpha-galactosyl immunoglobulin G (anti-Gal) is a natural antibody present in unusually high amounts in human sera. It constitutes as much as 1% of circulating immunoglobulin G in humans and displays a distinct specificity for the carbohydrate epitope galactosyl alpha(1----3) galactosyl (Gal alpha 1----3Gal). Recently, it has been suggested by various investigators that anti-Gal may be related to some autoimmune phenomena, since marked elevation of its titer was found in sera of patients with autoimmune thyroid disorders, rheumatoid arthritis, glomerulonephritis, and Chagas' disease. In view of the ubiquitous presence of anti-Gal in high titers in humans, throughout life, we hypothesized that, analogous with synthesis of anti-blood group antibodies against bacterial antigens, bacteria within normal intestinal flora may provide constant antigenic stimulation for the synthesis of anti-Gal. This hypothesis would imply that anti-Gal may bind to a variety of bacterial strains of human flora. In the present study, the interaction between affinity chromatography-purified anti-Gal and various bacterial strains was studied. By the use of a direct immunostaining assay and an enzyme-linked immunosorbent assay, anti-Gal was found to interact with a variety of Escherichia coli, Klebsiella, and Salmonella strains, some of which were isolates from normal stool. Furthermore, the anti-Gal-binding sites in some strains were found to be present on the carbohydrate portion of bacterial lipopolysaccharides. It is thus suggested that Gal alpha 1----3Gal epitopes in the outer membranes of normal flora enterobacteria may provide a continuous source for antigenic stimulation. Since there is no immune tolerance to the Gal alpha 1----3Gal carbohydrate structure in humans, anti-Gal seems to be constantly produced in response to these enterobacteria. In addition, bacteria which express Gal alpha----3Gal epitopes and which may adhere to various cells mediated binding of anti-Gal to human cell lines. These findings raise the possibility that anti-Gal may damage normal human tissues via inflammatory processes facilitated by bacterial Gal alpha 1----3Gal epitopes.