A series of compounds derived from 3-alkyl-N-hydroxysuccinimide have been synthesized and their inhibitory activity toward human leukocyte elastase has been investigated. Compounds having an isobutyl or isopropyl group at the C-3 position have been found to be particularly effective inactivators of the enzyme. The introduction of a trans-styryl group (as in compounds 16 and 18) results in a drastic enhancement in inhibitory activity indicative of a favorable interaction between the phenyl ring and the S2' subsite of the enzyme. The compounds were found to be highly stable in buffer solution with no apparent change in structural integrity after 17 h (the period of observation). Studies with model compounds and high-field NMR indicate that these compounds function as mechanism-based inhibitors of the enzyme. Porcine pancreatic elastase is not inhibited by these compounds, while chymotrypsin and human leukocyte cathepsin G are also efficiently inactivated.