Thrombin, usually regarded as a trypsin-like enzyme capable of hydrolyzing only esters of the amino acids arginine and lysine, was found to hydrolyze certain other amino acid esters which are considered specific chymotrypsin substrates. L-Phenylalanine methyl ester inhibited the activation of purified bovine prothrombin by autoprothrombin C, Ac-globulin, phospholipid, and calcium. It was subsequently shown that thrombin is capable of hydrolyzing L-phenylalanine methyl ester or L-tyrosine ethyl ester. This activity developed simultaneously with fibrogen clotting activity during activation of purified bovine prothrombin in 25% sodium citrate solutions. Moreover, the activity was closely associated with clotting activity on subsequent chromatography on DEAE-cellulose and Amberlite IRC-50 resin columns. All preparations of bovine thrombin, representing a wide range of purity, which have been examined, exhibited this hydrolyzing activity toward L-phenylalanine methyl ester. Further evidence linking this esterase activity with fibrinogen clotting activity was obtained when both activities were inhibited by 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate, an inhibitor of chymotrypsin.