To overcome the antibiotic resistance mechanism mediated by beta-lactamases, small-molecule beta-lactamase inhibitors, such as clavulanic acid, have been used. This approach, however, has applied selective pressure for mutations that result in beta-lactamases no longer sensitive to beta-lactamase inhibitors. On the basis of the structure of beta-lactamase inhibitor protein (BLIP), novel peptide inhibitors of beta-lactamase have been constructed. BLIP is a 165-amino-acid protein that is a potent inhibitor of TEM-1 beta-lactamase (K(i) = 0.3 nM). The cocrystal structure of TEM-1 beta-lactamase and BLIP indicates that residues 46 to 51 of BLIP make critical interactions with the active site of TEM-1 beta-lactamase. A peptide containing this six-residue region of BLIP was found to retain sufficient binding energy to interact with TEM-1 beta-lactamase. Inhibition assays with the BLIP peptide reveal that, in addition to inhibiting TEM-1 beta-lactamase, the peptide also inhibits a class A beta-lactamase and a class C beta-lactamase that are not inhibited by BLIP. The crystal structures of class A and C beta-lactamases and two penicillin-binding proteins (PBPs) reveal that the enzymes have similar three-dimensional structures in the vicinity of the active site. This similarity suggests that the BLIP peptide inhibitor may have a broad range of activity that can be used to develop novel small-molecule inhibitors of various classes of beta-lactamases and PBPs.