3-Oxo-β-sultams are both β-sultams and β-lactams and are a novel class of time-dependent inhibitors of elastase. The inhibition involves formation of a covalent enzyme-inhibitor adduct with transient stability by acylation of the active-site serine resulting from substitution at the carbonyl centre of the 3-oxo-β-sultam, C-N fission, and expulsion of the sulfonamide. The lead compound, N-benzyl-4,4-dimethyl-3-oxo-β-sultam 1 is a reasonably potent inhibitor against porcine pancreatic elastase with a second-order rate constant of 768 M-1 s-1 at pH 6, but also possesses high chemical reactivity with a half-life for hydrolysis of only 6 mins at the same pH in water. Interestingly, the hydrolysis of 3-oxo-β-sultams occurs at the sulfonyl centre with S-N fission and expulsion of the amide leaving group, whereas the enzyme reaction occurs at the acyl centre. Increasing selectivity between these two reactive centres was explored by examining the effect of substituents on the reactivity of 3-oxo-β-sultam towards hydrolysis and enzyme inhibition. The inhibition activity against porcine pancreatic elastase has a much higher sensitivity to substituent variation than does the rate of alkaline hydrolysis. A difference of 2000-fold is observed in the second-order rate constants, ki, for inhibition whereas there is only a 100-fold difference in the second-order rate constants, kOH, for alkaline hydrolysis within the series. The higher sensitivity of enzyme inhibition to substituents than that of simple chemical reactivity indicates a significant degree of molecular recognition of the 3-oxo-β-sultams by the enzyme.