This study provides evidence that pyelonephritogenic strains of Escherichia coli, which do not produce soluble hemolysin and possess mannose-resistant hemagglutinating activity, are able to adhere and penetrate to HEp-2 cells. Invasion and intracellular survival were analyzed by transmission electron microscopy and by viable counts after killing of extracellular bacteria by gentamicin. Cytochalasin D, which blocks polymerization of G-actin, markedly reduced the entry of E. coli into the cells and inhibited intracellular mobility of the bacteria. By using indirect fluorescent staining with anti-actin rabbit serum, direct evidence was obtained that interaction with the cytoskeleton of HEp-2 cells is necessary for invasion.