Approximately 35 000 Staphylococcus aureus surgical wound infections occur annually in the United States. To investigate why S aureus causes infection despite the perioperative administration of cephalosporins, we compared 35 methicillin-susceptible isolates recovered from deep wound infections that complicated cefazolin prophylaxis (18 of 1650 patients) and cefamandole prophylaxis (17 of 3702 patients) with 64 colonizing isolates from presurgical patients. Compared with both colonizing and cefamandole-associated isolates, S aureus isolates from cefazolin-associated infections were more resistant to cefazolin by specialized assays. Staphylococcus aureus isolates that produced the A and C variants of staphylococcal β-lactamase were associated with infections following cefazolin and cefamandole prophylaxis, respectively. These isolates hydrolyze the respective cephalosporins rapidly, suggesting that staphylococcal survival after perioperative prophylaxis may be mediated by in vivo degradation of the prophylactically administered cephalosporin. These data indicate that some S aureus wound infections occur because of deficiencies in antimicrobial effectiveness that are not detectable by routine susceptibility tests. This finding has important implications for the therapy and prevention of S aureus infection.