Clinical trials in surgery suggest that some failures of antibiotic prophylaxis are related to the in vivo degradation of beta-lactams by Staphylococcus aureus beta-lactamase. To explore this issue further, isogeneic isolates of S. aureus differing only in whether they contained the structural gene for type A staphylococcal beta-lactamase were constructed and compared for their ability to establish an abscess in a guinea pig model. With ampicillin prophylaxis, the ID50 was 870 cfu for the beta-lactamase-negative isolate VK7114 and 240 cfu for the beta-lactamase-producing isolate VK7115 (P < .001). Similarly, the ID50 was greater for the beta-lactamase-negative isolate when cefazolin prophylaxis was administered (599 vs. 128 cfu, VK7114 and VK7115; P < .001). In the setting of prophylaxis with beta-lactamase-susceptible antibiotics, beta-lactamase contributes to the pathogenesis of S. aureus wound infections.