A satisfactory mouse model of ascending urinary tract infection (UTI) must avoid inoculation-induced vesicoureteral reflex (VUR) yet still produce kidney and bladder infection in a substantial proportion of mice. To define inoculation conditions that would satisfy both these conditions, mice were evaluated for VUR immediately after inoculation under a variety of conditions and were assessed for kidney and bladder infection 48 h after inoculation. Elimination of VUR required a slowed infusion rate, a reduced inoculum volume (25 microliter), and use of less traumatic methods for euthanasia and organ harvest. Bladder and kidney infection were highly prevalent at 48 h among mice inoculated under VUR-free conditions with either of 2 wild type Escherichia coli strains. Together with reports from other investigators, these findings indicate that satisfactory experimental conditions for the mouse model of ascending UTI can be defined empirically but may be laboratory-specific.