Clavulanic acid is a potent mechanism-based inhibitor of TEM-1 and SHV-1beta-lactamases, enzymes that confer resistance to beta-lactams in many gram-negative pathogens. This compound has enjoyed widespread clinical use as part of beta-lactam beta-lactamase inhibitor therapy directed against penicillin-resistant pathogens. Unfortunately, the emergence of clavulanic acid-resistant variants of TEM-1 and SHV-1 beta-lactamase significantly compromise the efficacy of this combination. A single amino acid change at Ambler position Ser130 (Ser --> Gly) results in resistance to inactivation by clavulanate in the SHV-1 and TEM-1beta-lactamases. Herein, we investigated the inactivation of SHV-1 and the inhibitor-resistant S130G variant beta-lactamases by clavulanate. Using liquid chromatography electrospray ionization mass spectrometry, we detected multiple modified proteins when SHV-1 beta-lactamase is inactivated by clavulanate. Matrix-assisted laser desorption ionization-time of flight mass spectrometry was used to study tryptic digests of SHV-1 and S130Gbeta-lactamases (+/- inactivation with clavulanate) and identified peptides modified at the active site Ser70. Ultraviolet (UV) difference spectral studies comparing SHV-1 and S130Gbeta-lactamases inactivated by clavulanate showed that the formation of reaction intermediates with absorption maxima at 227 and 280 nm are diminished and delayed when S130Gbeta-lactamase is inactivated. We conclude that the clavulanic acid inhibition of the S130G beta-lactamase must follow a branch of the normal inactivation pathway. These findings highlight the importance of understanding the intermediates formed in the inactivation process of inhibitor-resistant beta-lactamases and suggest how strategic chemical design can lead to novel ways to inhibit beta-lactamases.