Phenyl esters of 2-benzoylbenzoates were determined to be inhibitors of the serine protease enzymes chymotrypsin and thrombin. Thus, p-guanidinophenyl 2-benzoylbenzoate (Ib) inhibited thrombin while the corresponding p-nitrophenyl ester (la) inhibited chymotrypsin activity. Other p-nitrophenyl esters were prepared that display activity as chymotrypsin inhibitors, three having methoxy group substitution on the benzoyl ring: 2-methoxy (2a), 2,5-dimethoxy (3a), and 2,4,5-trimethoxybenzoyl (4). After incubation with Ib, an acyl thrombin was isolated that showed no lytic activity but which slowly regained activity in pH 7.4 buffer. Irradiation of this acyl enzyme with 366 nm light led to an enzyme that showed no gain of lytic activity over time. Incubation of chymotrypsin with 1a-3a and 4 led to acyl enzymes which showed no activity but which regained activity slowly. Irradiation of these inactive acyl enzymes with 366 nm light led to a rapid increase in enzyme activity. The formation of acylchymotrypsins that can be photochemically deacylated is suggested by these data. Experiments that relate to the mechanism of enzyme acylation and the subsequent photochemistry of the acylenzymes are reported.