A series of 2-pyranones, bearing halogens or electron-withdrawing groups at the 6-position and alkyl, aryl, or aralkyl groups at positions 3, 4, and 5, were synthesized to investigate their binding to and inactivation of chymotrypsin. Both binding and inactivation by 2-pyranones are sensitive to substitutions on positions 3, 4, 5, and 6. Binding was poorest with alkyl substituents on position 3 and best with phenyl substitution, with benzyl or benzyl-like substitution falling in between. The sequence of binding of 6-substituted pyrones is Cl greater than Br greater than H greater than CF3. 6-Chloro-2-pyranones bearing 4-phenyl or 3-(2-naphthylmethyl) substituents effected rapid inactivation of chymotrypsin, while those having 3-benzyl or 3-(1-naphthylmethyl) substituents gave slow inactivation and those with 3-phenyl or 3-alkyl substituents gave no inactivation. Only the 6-halopyrones demonstrated inactivation, with chloro-substituted ones acting faster than bromo-substituted ones.