The CS31A antigen was first described for septicemic and enterotoxigenic bovine E. coli strains. In our study, of 597 human Escherichia coli strains isolated from diarrheagenic stools of hospitalized patients, 30 (5%) hybridized with the CS31A DNA probe. These CS31A-positive E. coli strains diffusely adhered to Caco-2 and/or HEp-2 cells and produced a major surface protein of either 30 or 30.5 kDa according to the strain. These proteins were antigenically related to the two forms of the CS31A antigen, namely, CS31A-L and CS31A-H. Genes encoding CS31A were located on 140-kb conjugative R plasmids. E. coli transconjugants expressed major surface proteins similar to those of the wild-type strains and adhered to Caco-2 and/or HEp-2 cells. An association of CS31A and another adhesive factor of the Dr family was found in 70% of wild-type strains, since 21 strains hybridized with the diffuse adhesion DNA probe corresponding to the accessory gene (daaC) of the F1845 adhesin. Comparison of the restriction patterns of the 140-kb R plasmids of the CS31A-positive E. coli strains showed these plasmids to be similar. Hybridization experiments indicated that the genes encoding CS31A and resistance to penicillin were located together on either of two 20- or 27-kb EcoRI restriction fragments in four E. coli strains. We reported a similar linkage between these genes in Klebsiella pneumoniae strains which produced CF29K, a CS31A-like antigen. These results suggest a horizontal transfer between E. coli and K. pneumoniae strains.