The role of a plasmid in the virulence activity of an enteropathogenic Escherichia coli (EPEC) strain belonging to serotype 0111:NM was examined. EPEC strain B171, which is resistant to chloramphenicol, streptomycin, sulfathiazole, and tetracycline, harbors a 54-megadalton plasmid, pYR111, and exhibits localized adherence (LA) with HeLa cells. Curing the plasmid yielded strain B171-4, which had lost the ability to exhibit LA, resistance to the antibiotics, and the lipopolysaccharide (LPS) O-antigenic polysaccharide. To confirm that these phenotypic characteristics were specified by pYR111, the plasmid was transferred by conjugation into a nalidixic acid-resistant strain of E. coli HB101. LA and antimicrobial resistance were expressed in most of the transconjugants examined. The O-polysaccharide side chains, antigenically reactive with O111-specific antiserum, were also expressed by the transconjugants. Although EPEC plasmids coding for both drug resistance and LA have been described, an EPEC plasmid encoding the expression of an LPS O antigen has not been previously reported. Similar findings described for some Shigella and Salmonella strains suggest that plasmid-encoded modification of the LPS in some enteric bacterial species may be more common than previously recognized and may contribute to the characteristic virulence activity of the organism.