Infection of humans with verotoxin-producing Escherichia coli (VTEC) O113:H21 is associated with clinical features comparable to those associated with infection with attaching and effacing VTEC strains including those of serotype O157:H7. We have shown previously that the adhesion phenotype of VTEC O157:H7 is influenced by the presence of a homolog of the chromosomal eaeA (for E. coli attaching and effacing) gene. In contrast, by colony blot hybridization, VTEC O113:H21 is negative for the eaeA gene. Therefore, the aim of this study was to define the adhesion phenotype of VTEC O113:H21 strain CL-15 to both cultured epithelial cells (HEp-2) and rabbit intestine in vivo. Under transmission electron microscopy, areas of microvillus effacement were observed in regions directly beneath the organism in CL-15-infected cells both in vitro and in vivo. However, F-actin adhesion pedestals on the host plasma membrane were absent. Failure of CL-15 to induce polymerization of actin was confirmed by using staining of F-actin with fluorescein-labeled phalloidin. Under indirect immunofluorescence microscopy, CL-15-infected HEp-2 cells also failed to demonstrate the recruitment of another cytoskeletal element, alpha-actinin, below foci of bacterial adhesion. In contrast, VTEC O157:H7 infection of HEp-2 cells was associated with increased alpha-actinin immunofluorescence. These findings suggest that bacterial factors distinct from those of EaeA are necessary for the adhesion phenotype of VTEC O113:H21.