A gentamicin survival assay, using primary human renal epithelial cells and Escherichia coli strains isolated from the feces of asymptomatic individuals and from the urine or blood of patients with acute pyelonephritis, was used to investigate bacterial internalization as a model for renal parenchymal invasion in pyelonephritis. E. coli strains, regardless of their origin, efficiently entered into human renal epithelial cells, a process inhibited by cytochalasin D. While the percentage of survival of nonhemolytic pyelonephritis isolates did not differ from that of fecal isolates, survival of hemolytic pyelonephritis strains was lower than that of nonhemolytic strains, perhaps as a consequence of the greater cytotoxicity of hemolytic strains. There was no evidence of intracellular multiplication of E. coli. These results demonstrate that human renal epithelial cells are capable of efficient uptake of E. coli regardless of the source of the bacteria.