Infection of HEp-2 cells by enteropathogenic Escherichia coli (EPEC) was examined by transmission and scanning electronmicroscopy. EPEC strains of serogroups O111:K58 and O55:K59 recently isolated from human patients did not exhibit enterotoxic activity, as judged by the Vero-cell and suckling-mouse assays, or invasive ability as judged by the Sereny test. These strains attached to and penetrated HEp-2 cells. Transmission electronmicroscopy showed bacteria in close contact with cell membranes 15 min after infection; later, intense swelling and budding of membranes and penetration of EPEC into the cell cytoplasm occurred. Intracellular bacteria were enclosed in membrane-bound vacuoles in the cell cytoplasm underlying localised adherence sites observed by light microscopy. Scanning electronmicroscopy showed morphologically altered membranes only at the sites of bacterial attachment. Bacteria inactivated by ultraviolet light were not internalised and cytochalasin B (greater than or equal to 10 mg/L) markedly inhibited uptake. These observations suggest that penetration of EPEC into HEp-2 cells occurs by an endocytic process in metabolically active bacteria.