Proteus mirabilis, a gram-negative bacterium, is a frequent cause of complicated urinary tract infections in those with functional or anatomical abnormalities or those subject to long-term catheterization. To systematically identify surface-exposed antigens as potential vaccine candidates, proteins in the outer membrane fraction of bacteria were separated by two-dimensional gel electrophoresis and subjected to Western blotting with sera from mice experimentally infected with P. mirabilis. Protein spots reactive with sera were identified by mass spectrometry, which in conjunction with the newly completed genome sequence of P. mirabilis HI4320, was used to identify surface-exposed antigens. Culture conditions that may mimic in vivo conditions more closely than Luria broth (growth in human urine and under iron limitation and osmotic stress) were also used. Thirty-seven antigens to which a humoral response had been mounted, including 23 outer membrane proteins, were identified. These antigens are presumably expressed during urinary tract infection. Protein targets that are both actively required for virulence and antigenic may serve as protective antigens for vaccination; thus, five representative antigens were selected for use in virulence studies. Strains of P. mirabilis with mutations in three of the corresponding genes (the PMI0047 gene, rafY, and fadL) were not attenuated in the murine model of urinary tract infection. Putative iron acquisition proteins PMI0842 and PMI2596, however, both contribute to fitness in the urinary tract and thus emerge as vaccine candidates.