Clostridium difficile is a leading cause of nosocomial infection in the developed world, causing antibiotic-associated disease in susceptible populations. The identity of immunogenic proteins is important in understanding the pathogenesis of disease and in the design of vaccines. This study analysed the sera of six patients infected during a hospital outbreak of a C. difficile ribotype 017 strain. Using a proteomics-based approach, cell wall proteins were separated by two-dimensional PAGE, and immunoreactive proteins were revealed by reaction with patient sera. The identity of immunoreactive proteins was established by MS. Forty-two different proteins were identified in total. All patient sera reacted with at least one component of the surface-layer protein (SLP), four reacted with both components (high- and low-molecular-mass SLPs), and five reacted with one other cell wall protein, suggesting that these are immunodominant antigens. The role of these proteins as potential vaccine candidates and their roles in pathogenesis deserve further study.