A series of fluorinated alpha-keto acid derivatives [PhCHFCOCO2R,PhCH2CHFCOCO2R,PhCF2-COCO2R, and PhCH2CF2COCO2R (R = H, Me, and Et)] was synthesized. They were inhibitors of chymotrypsin, with Ki values ranging from 4700 to 15 microM. Benzylpyruvic derivatives were generally more potent than the corresponding phenylpyruvic analogs. Esters of the first series were also more potent than their corresponding acids, and potency increased with the number of fluorine atoms. By replacing the ethoxy group of PhCH2CF2COCO2Et (15b) with an amino acid chain (i.e., alanyl-leucyl-arginine methyl ester hydrochloride and alanyl-leucyl-valine ethyl ester), the resultant peptides PhCH2CF2COCO-Ala-Leu-Arg-OMe.HCl.H2O (20) and PhCH2CF2COCO-Ala-Leu-Val-OEt.H2O (23) were found to be slow-binding inhibitors of chymotrypsin with considerably lower Ki values (0.19 and 3.6 microM, respectively). 19F NMR studies indicate, in the case of 20, the presence of an enzyme-inhibitor complex with a hemiketal structure similar to those observed between trifluoromethyl ketones and chymotrypsin.