β-Sultams are the sulfonyl analogues of β-lactams, and N-acyl β-sultams are novel inactivators of the class C β-lactamase of Enterobacter cloacae P99. They sulfonylate the active site serine residue to form a sulfonate ester which subsequently undergoes C−O bond fission and formation of a dehydroalanine residue by elimination of the sulfonate anion as shown by electrospray ionization mass spectroscopy. The analogous N-acyl β-lactams are substrates for β-lactamase and undergo enzyme-catalyzed hydrolysis presumably by the normal acylation−deacylation process. The rates of acylation of the enzyme by the β-lactams, measured by the second-order rate constant for hydrolysis, kcat/Km, and those of sulfonylation by the β-sultams, measured by the second-order rate constant for inactivation, ki, both show a similar pH dependence to that exhibited by the β-lactamase-catalyzed hydrolysis of β-lactam antibiotics. Electron-withdrawing groups in the aryl residue of the leaving group of N-aroyl β-lactams increase the rate of alkaline hydrolysis and give a Bronsted βlg of −0.55, indicative of a late transition state for rate-limiting formation of the tetrahedral intermediate. Interestingly, the corresponding Bronsted βlg for the β-lactamase-catalyzed hydrolysis of the same substrates is −0.06, indicative of an earlier transition state for the enzyme-catalyzed reaction. By contrast, although the Bronsted βlg for the alkaline hydrolysis of N-aroyl β-sultams is −0.73, similar to that for the β-lactams, that for the sulfonylation of β-lactamase by these compounds is −1.46, compatible with significant amide anion expulsion/S−N fission in the transition state. In this case, the enzyme reaction displays a later transition state compared with hydroxide-ion-catalyzed hydrolysis of the β-sultam.