beta-Lactamases are bacterial enzymes that hydrolyze beta-lactam antibiotics to render them inactive. The beta-lactamase inhibitor protein (BLIP) of Streptomyces clavuligerus, is a potent inhibitor of several beta-lactamases, including the TEM-1 enzyme (Ki = 0.6 nM). Evidence from the TEM-1/BLIP co-crystal suggests that two BLIP residues, Asp-49 and Phe-142, mimic interactions made by penicillin G when bound in the active site of TEM-1. To determine the importance of these two residues, a heterologous expression system for BLIP was established in Escherichia coli. Site-directed mutagenesis was used to change Asp-49 and Phe-142 to alanine, and inhibition constants (Ki) for both mutants were determined. Each mutation increases the Ki for BLIP inhibition of TEM-1 beta-lactamase approximately 100-fold. To address how these two positions effect the specificity of beta-lactamase binding, Ki values were determined for the interaction of wild-type BLIP, as well as the D49A and F142A mutants, with two extended spectrum beta-lactamases (the G238S and the E104K TEM variants). Positions 104 and 238 are located in the BLIP/beta-lactamase interface. Interestingly, the three BLIP proteins inhibited the G238S beta-lactamase mutant to the same degree that they inhibited TEM-1. However, wild-type BLIP has a higher Ki for the E104K beta-lactamase mutant, suggesting that interactions between BLIP and beta-lactamase residue Glu-104 are important for wild-type levels of BLIP inhibition.