A series of model peptides containing a-trifluoromethyl-substituted amino acids in five different positions relative to the predominant cleavage site of the serine protease a-chymotrypsin was synthesized by solution methods to investigate the influence of a-Tfm substitution on the proteolytic stability of peptides. Proteolysis studies demonstrated absolute stability of peptides substituted in the P 1 position and still considerable proteolytic stability for peptides substituted at the P 2 and P 0 2 positions compared with the corresponding unsubstituted model peptide. Comparison with peptides containing the fluorine-free disubstituted amino acid a-aminoisobutyric acid allowed to separate electronic from steric effects. Furthermore, the absolute configuration of the a-Tfm-substituted amino acid was found to exert considerable effects on the proteolytic stability, especially in P 0 1 substituted peptides. Investigations of this phenomenon using empirical force field calculations revealed that in the (S,R,S)-diasteromer the steric constraints exhibited by the a-Tfm group can be outweighed by an advantageous interaction of the fluorine atoms with the serine side chain of the enzyme. In contrast, a favourable interaction between substrate and enzyme is impossible for the (S,S,S)-diastereomer.