A novel and functional conjugative transfer system identified in O119:H2 enteropathogenic Escherichia coli (EPEC) strain MB80 by subtractive hybridization is encoded on a large multidrug resistance plasmid, distinct from the well-described EPEC adherence factor (EAF) plasmid. Variants of the MB80 conjugative resistance plasmid were identified in other EPEC strains, including the prototypical O111:NM strain B171, from which the EAF plasmid has been sequenced. This separate large plasmid and the selective advantage that it confers in the antibiotic era have been overlooked because it comigrates with the virulence plasmid on conventional gels.