Ten analogs of the peptide A-Phe(NO2)-Phe-Val-Leu-B were synthesized and tested as substrates for cathepsin D and pepsin. The best substrate found for cathepsin D, Phe-Ala-Ala-Phe(NO2)-Phe-Val-Leu-OM4P (kcat = 2.9 s-1; Km = 7.1 microM), has the largest kcat/Km value (408 mM-1 s-1) reported to date for this enzyme. The effect of peptide structure on solubility and kinetic parameters is discussed. The peptide provides a useful new substrate for continuous assay of cathepsin D.