Roles of the ZnuACB and ZupT transporters were assessed in Escherichia coli K-12 and uropathogenic E. coli (UPEC) CFT073. K-12 and CFT073 Deltaznu DeltazupT mutants demonstrated decreased (65)Zn(2+) uptake and growth in minimal medium. CFT073Deltaznu demonstrated an intermediate decrease of (65)Zn(2+) uptake and growth in minimal medium, whereas the CFT073DeltazupT mutant grew as well as CFT073 and exhibited a less marked decrease in (65)Zn(2+) uptake. CFT073 mutants grew as well as the wild type in human urine. In competitive infections in CBA/J mice, the DeltazupT mutant demonstrated no disadvantage during urinary tract infection. In contrast, the UPEC Deltaznu and Deltaznu DeltazupT strains demonstrated significantly reduced numbers in the bladders (mean 4.4- and 30-fold reductions, respectively) and kidneys (mean 41- and 48-fold reductions, respectively). In addition, in single-strain infection experiments, the Deltaznu and Deltaznu DeltazupT mutants were reduced in the kidneys (P = 0.0012 and P < 0.0001, respectively). Complementation of the CFT073 Deltaznu DeltazupT mutant with the znuACB genes restored growth in Zn-deficient medium and bacterial numbers in the bladder and kidneys. The loss of the zinc transport systems decreased both motility and resistance to hydrogen peroxide, which could be restored by supplementation with zinc. Overall, the results indicate that Znu and ZupT are required for growth in zinc limited-conditions, that Znu is the predominant zinc transporter, and that the loss of Znu and ZupT has a cumulative effect on fitness during UTI, which may in part be due to reduced resistance to oxidative stress and motility.