Chromophore substrates Dnp-Ala-Glu-Phe-Ala-Arg-NH2 and Dnp-Ala-Ala-Phe-Nle-Ala-Arg-NH2 of aspartic proteases were synthesized by a combination of chemical and enzymic methods. The kinetic parameters of their hydrolysis with pepsin, aspergyllopepsin, and chymosin were determined. The introduction of Nle in the P1' position gives stable enzyme-substrate complexes with pepsin and chymosin. A Glu residue at the P2 position contributes significantly to an increase in kcat for the chymosin hydrolysis.