A DNA segment located immediately upstream of the eaeA gene of enterohemorrhagic Escherichia coli O157:H7 strain HA1 was cloned and sequenced. This segment contained an open reading frame encoding a predicted protein of 156 amino acids. A database search identified similar open reading frames upstream of the eaeA gene in two other bacterial pathogens, i.e. enteropathogenic E. coli and Citrobacter freundii. The predicted amino acid sequence of the enterohemorrhagic E. coli protein shared 96.8% and 94.2% identity with the enteropathogenic E. coli and C. freundii sequences, respectively. Because the open reading frame is located within the locus of enterocyte effacement region of the E. coli chromosome, a 'hot spot' for insertion of virulence factor genes, and shares high sequence homology with attaching and effacing EPEC and C. freundii, this protein may be associated with pathogenicity of E. coli O157:H7.