The interaction of serine protease (esterases) with 6-chloro-2-pyrones was investigated. Time-dependent inactivation of chymotrypsin, .alpha.-lytic protease, pig liver elastase and cholinesterase was found with 3- and 5-benzyl-6-chloro-2-pyrone, as well as 3- and 5-methyl-6-chloro-2-pyrone. No inactivation was observed with the unsubstituted 6-chloro-2-pyrone. The substituted pyrones did not inactivate papain or carboxypeptidase A, as well as a number of other nonproteolytic enzymes. The substituted chloropyrones, therefore show considerable selectivity toward serine proteases. Analogs in which the 6-chloro substituent is replaced by H or OH do not inactivate. The presence of the halogen is therefore, essential for inactivation. Chymotrypsin catalyzes the hydrolysis of 3-benzyl-6-chloro-2-pyrone. At pH 7.5 (E)-4-benzyl-2-pentenedioic acid is the major product, and 2-benzyl-2-pentenedioic anhydride is a minor product. The ratio of hydrolysis product found to the number of enzyme molecules inactivated varies from 14 to 40. The enzyme inactivated with the 3-benzyl compound does not show a spectrum characteristic of the pyrone ring. This suggests that inactivation by 3-benzyl-6-chloro-2-pyrone occurs in a mechanism-based fashion after enzymatic lactone hydrolysis. When the enzyme is inactivated with the 5-benzyl compound, absorbance due to the pyrone ring is observed. Inactivation may occur through an active site directed mechanism involving a 1,6-conjugate addition of an active site nucleophile to the pyrone ring.