Uncomplicated urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) represents a prevalent and potentially severe infectious disease. In this study, we describe the application of an immunoproteomics approach to vaccine development that has been used successfully to identify vaccine targets in other pathogenic bacteria. Outer membranes were isolated from pyelonephritis strain E. coli CFT073 cultured under conditions that mimic the urinary tract environment, including iron limitation, osmotic stress, human urine, and exposure to uroepithelial cells. To identify antigens that elicit a humoral response during experimental UTI, outer membrane proteins were separated by two-dimensional gel electrophoresis and probed using pooled antisera from 20 CBA/J mice chronically infected with E. coli CFT073. In total, 23 outer membrane antigens, including a novel iron compound receptor, reacted with the antisera and were identified by mass spectrometry. These antigens also included proteins with known roles in UPEC pathogenesis, such as ChuA, IroN, IreA, Iha, IutA, and FliC. These data demonstrate that an antibody response is directed against these virulence-associated factors during UTI. We also show that the genes encoding ChuA, IroN, hypothetical protein c2482, and IutA are significantly more prevalent (P < 0.01) among UPEC strains than among fecal-commensal E. coli isolates. Thus, we suggest that the conserved outer membrane antigens identified in this study could be rational candidates for a UTI vaccine designed to elicit protective immunity against UPEC infection.