Most of the Staphylococcus aureus virulence factors are either cell surface exposed or secreted. Here we report a global and quantitative analysis of staphylococcal cell surface-associated proteins using a combination of (14)N(15)N metabolic labeling, biotinylation, and GeLC-MS/MS. To address the important question of S. aureus pathophysiology, we analyzed the influence of the alternative sigma factor sigma(B) on the expression of cell surface-associated proteins. Therefore, we compared the methicillin-resistant S. aureus wild-type strain COL with its sigB mutant, because sigma(B) might play a crucial role in the pattern of the surface proteome. A total of 296 proteins from growing and nongrowing cells could be quantified. One third of these proteins are known as cell surface-associated, including 3 sortase substrates, 3 cell wall-associated proteins, 35 lipo-, 23 membrane-, and 34 signal peptide-containing proteins comparing wild-type and sigB mutant. Fourty nine surface-associated proteins were modulated by sigma(B), including 21 proteins already known to be SigB-dependent or SigB-influenced. More proteins were down- (31 proteins) than up-regulated (18 proteins) in the sigB mutant. Our approach revealed 28 surface-associated proteins not previously reported as SigB-dependent or -influenced, expanding the group of surface-associated proteins and virulence factors modulated by SigB.