Enteropathogenic Escherichia coli (EPEC) is capable of attaching intimately to epithelial cells and effacing their microvilli. A chromosomal locus, eaeA (originally eae), is required for the intimate attachment aspect of this effect. We report the mapping of a region of the EPEC chromosome that is located immediately downstream of the eaeA gene and that is also necessary for intimate attachment. An isogenic in-frame deletion mutation in one of the open reading frames identified in this region was engineered. Because the resulting mutant, like an eaeA deletion mutant, is deficient in the ability to attach intimately to epithelial cells, the mutated gene is designated eaeB. Full activity is restored to the eaeB mutant when the cloned gene is reintroduced on a plasmid. The eaeB mutant remains capable of producing intimin, the product of the eaeA gene. No differences in the fractionation properties or electrophoretic mobility of intimin are apparent in the eaeB mutant. The product of the eaeB locus was identified by in vitro transcription-translation. The nucleotide sequence of the eaeB gene predicts a protein that contains a sequence motif common to several aminotransferase enzymes. These results indicate that the attaching and effacing effect is a complex phenotype dependent on a gene cluster present on the EPEC chromosome.