Escherichia coli Dr adhesin and decay-accelerating factor (DAF) receptor-mediated interaction was proposed as the mechanism of ascending urinary tract infection (UTI) and chronic interstitial nephritis. This report provides novel evidence for Dr fimbriae operon-mediated invasive capacity of Dr+ E. coli. Insertional mutants draE, draC, and draB, and adherent draD and UV-inactivated BN406 were unable to enter HeLa cells. Complementation of the dra mutation restored invasiveness. Internalization was inhibited by anti-Dr fimbriae IgG (100%), anti-SCR-3 domain of DAF (75%), and nocodazole (95%). Increased receptor-ligand density occurred at the site of internalization. Internalized Dr+ E. coli did not significantly multiply in the HeLa cell line. Accordingly, the dra operon and DAF were required for microtubule-dependent internalization of E. coli to HeLa cells. The relatively low invasion and multiplication rates of Dr+ E. coli may hypothetically contribute to the postattachment steps of ascending UTI and chronic renal infection.