In order to investigate the ability of enteropathogenic Escherichia coli (EPEC) to invade epithelial cells, 24 strains of diarrhea-causing E. coli were studied with a HEp-2 cell-gentamicin invasion assay. Invasive ability was expressed as the percentage of the inoculum surviving gentamicin after incubation of bacteria with HEp-2 cells. Geometric mean survival of EPEC strains possessing the EPEC adherence factor (EAF+ EPEC) was 5.177%, which was significantly greater than survival of enteroinvasive E. coli (EIEC) strains (1.871%). EPEC strains lacking EAF (EAF-EPEC), enterotoxigenic E. coli (ETEC), and enterohemorrhagic E. coli (EHEC) were significantly less invasive (geometric mean survival, 0.032%, 0.013%, and 0.009%, respectively). The variation in bacterial recovery was not due to differences in the number of HEp-2 cells remaining attached to the plates, as measured by the retention of crystal violet stain in parallel assays. Transmission electron microscopy confirmed the presence of many intracellular EAF+ EPEC and EIEC, whereas EAF- EPEC, EHEC, and ETEC were found primarily outside the cells. Epithelial cell invasion is an overlooked property of EAF+ EPEC of potential relevance in disease pathogenesis.