The espA, espB and espD genes from enteropathogenic Escherichia coli were previously shown to be essential for triggering the signal transduction in infected host cells. We have cloned and determined the nucleotide sequences of the espA, espB and espD homologues from an E. coli strain (4221) isolated from a dog which manifested the attaching and effacing lesions in the small intestine. This strain is designated as a dog enteropathogenic E. coli. When comparing predicted amino acid sequences to those of the corresponding proteins from enteropathogenic E. coli O127, enterohemorrhagic E. coli serotype O26, enterohemorrhagic E. coli O157 and rabbit enteropathogenic E. coli, the EspA hiig protein showed the same level of similarity (75% identity) with EspA of enteropathogenic E. coli O127 and rabbit enteropathogenic E. coli. The EspB hiig protein showed the highest similarity with the EspB of enteropathogenic E. coli O127 (99% identity). The EspD hiig protein showed 88% identity with the EspD iig . We constructed and purified a maltose-binding fusion protein containing the product of the entire espB hiig gene of the dog enteropathogenic E. coli strain 4221. Purified maltose-binding protein-EspB hiig fusion protein was shown to bind efficiently to HEp-2 cells in a localized fashion as shown by immunofluorescence microscopy. In addition, when the dog enteropathogenic E. coli strain 4221 was grown in tissue culture medium (DMEM) supplemented with serum, a secreted 36-kDa protein was identified by immunoblot analysis using a polyclonal antiserum against the maltosebinding protein-EspB hiig fusion protein. z