Hafnia alvei is an emerging human pathogen associated with sporadic cases and outbreaks of diarrhea. Bangladeshi isolates of H. alvei possess the Escherichia coli attaching and effacing (eaeA) gene and demonstrate an attaching and effacing phenotype. In the present study we examined 11 Canadian H. alvei isolates and strain 19,982 from Bangladesh to determine if the formation of attaching and effacing lesions is a property shared among multiple isolates. Attaching and effacing lesions were detected by induction of tyrosine kinase protein phosphorylation and cytoskeletal rearrangements in infected tissue culture epithelial cells with immunofluorescence microscopy and by the examination of infected cells with transmission electron microscopy. The presence of the eaeA gene was examined by PCR and colony blot hybridization. Profiles of outer membrane protein extracts, chromosomal macrorestriction fragments, and plasmids were also examined. Accumulation of host phosphotyrosine proteins and rearrangement of the cytoskeletal protein alpha-actinin were both observed in HEp-2 cells infected with H. alvei 19,982. In contrast, none of the other 11 clinical H. alvei isolates demonstrated either of these responses, nor did they form attaching and effacing lesions under electron microscopy. Consistent with the absence of the attaching and effacing phenotype, these clinical isolates did not possess the eaeA gene. The outer membrane protein profiles of all the Canadian isolates were identical but differed from that of H. alvei 19,982. Pulsed-field gel electrophoresis and plasmid profile analyses of the clinical H. alvei isolates differed substantially from those of the Bangladeshi strain. These results indicate that there is heterogeneity among H. alvei strains with respect to signal transduction, attaching and effacing adhesion, outer membrane constituents, and genotype. Epidemiological studies on enteropathogenic H. alvei thus need to go beyond simple species designations and require specific identification of the virulent clones.