We have attempted to characterize the binding specificity of the coli surface 3 (CS3) subcomponent of colonization factor antigen II of enterotoxigenic Escherichia coli, by means of an immunoblot method in which the binding of fimbriated bacteria to sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated rabbit intestinal cell membranes was evaluated. Isolated CS3 fibrillae as well as bacteria expressing CS3 on their surface bound to several intestinal cell membrane structures, i.e., structures present in the electrophoretic front and in the 30- to 35-kDa range and, most prominently, 120- to 140-kDa structures. Delipidization and protein digestion of the rabbit brush borders revealed that CS3 bound to structures of a proteinaceous nature. Sodium meta-periodate oxidation of the intestinal cell membranes abolished all their CS3 binding activity, indicating that CS3 bound to carbohydrate moieties of glycoproteins. The binding of CS3 to the separated intestinal proteins could also be inhibited by preincubation with the lectin derived from Maackia amurensis, indicating that CS3 bound to galactoproteins in the rabbit intestine. Inhibition experiments using equimolar amounts of various gangliosides demonstrated that GM1, asialo-GM1, and GM2 inhibited the binding of CS3 equally well, whereas GM3 was not as effective. These results suggested that the critical CS3 binding epitope consisted of the carbohydrate sequence GalNAcβ1-4Gal. This was supported by electron microscopic experiments showing that this disaccharide, O linked to bovine serum albumin via a spacer, localized around CS3-positive bacteria but not at all around corresponding CS3-negative mutants. Furthermore, CS3-expressing bacteria recognized this neoglycoprotein when it was immobilized on nitrocellulose. The GalNAcβ1-4Gal disaccharide has also been implicated as a binding structure for other pathogenic bacteria such as enteropathogenic E. coli and Pseudomonas aeruginosa.