The multiple antibiotic resistance of methicillin-resistant strains of Staphylococcus aureus (MRSA) has become a major clinical problem worldwide. The key determinant of the broad-spectrum β-lactam resistance in MRSA strains is the penicillin-binding protein 2a (PBP2a). Because of its low affinity for β-lactams, PBP2a provides transpeptidase activity to allow cell wall synthesis at β-lactam concentrations that inhibit the β-lactam-sensitive PBPs normally produced by S. aureus. The crystal structure of a soluble derivative of PBP2a has been determined to 1.8 Å resolution and provides the highest resolution structure for a high molecular mass PBP. Additionally, structures of the acyl-PBP complexes of PBP2a with nitrocefin, penicillin G and methicillin allow, for the first time, a comparison of an apo and acylated resistant PBP. An analysis of the PBP2a active site in these forms reveals the structural basis of its resistance and identifies features in newly developed β-lactams that are likely important for high affinity binding.