The Gram-positive bacterium S. aureus is one of the most common causes of contagious bovine mastitis. A predominant S. aureus clone persists within its host over a long period. In the present study, we compared the surface proteome of two isolates, collected from a naturally infected dairy cow with chronic, subclinical mastitis: an initial isolate (IN) and a host-adapted strain (HA). The isolates were grown under iron- and oxygen-limiting conditions in RPMI 1640 medium. Bacterial cells were isolated at the early stationary phase and used for preparation of surface-associated proteins by applying a surface-shaving and a surface-biotinylation approach. Proteins were identified by gelfree (surface-shaving approach) and GeLC-MS/MS (biotinylation approach) analyses.