The locus of enterocyte effacement (LEE) is necessary for enteropathogenic Escherichia coli to cause characteristic attaching and effacing lesions in host cells. To determine whether sequences at the extreme right end of the LEE downstream of the espB gene are required for attaching and effacing, we constructed a mutant with an Ω-interposon insertion immediately downstream of espB. This mutant is incapable of attaching and effacing, of secreting EspA and EspB and of inducing tyrosine phosphorylation of host cell proteins. These phenotypes are restored by a plasmid containing the extreme right end of the LEE. The nucleotide sequence of this region reveals a relatively low G+C content, remnants of transposons, and several open reading frames. The predicted products of these open reading frames include a potential chaperone, a potential component of the secretion apparatus, and a hypothetical peptide with proline rich repeats reminiscent of several eukaryotic proteins. These data indicate that the extreme right end of the LEE is required for attaching and effacing and reveal information relevant to the origin and function of the LEE.