Fluorogenic substrates for cathepsin D; A-Tyr-Phe(NO2)-Leu-Leu (A; Ala-Arg-Pro-Lys-Pro-Leu-Leu-, Arg-Pro-Lys-Pro-Leu-Leu-, Pro-Lys-Pro-Leu-Leu-, Lys-Pro-Leu-Leu-, Pro-Leu-Leu-) and B-Phe(NO2)-Tyr-Leu-Leu (B; Arg-Pro-Lys-Pro-Leu-Leu-, Pro-Lys-Pro-Leu-Leu-, Lys-Pro-Leu-Leu-, Pro-Leu-Leu-) (Phe(NO2), p-nitrophenylalanine) were synthesized and digested by cathepsin D and pepsin. The fluorescence at 303 nm (excitation at 260 nm) was increased with the hydrolysis of the substrates. The minimum detectable cathepsin D concentrations for these substrates were 0.5-4 nM and pepsin concentrations were 0.1-0.8 nM except Pro-Leu-Leu-Tyr-Phe(NO2)-Leu-Leu under the following conditions: substrate concentration, 20 μM; measuring time, 3 min. The hydrolysis rate constants (kcat/Km) of B-Phe(NO2)-Tyr-Leu-Leu for cathepsin D were same or 2-3 times greater than A-Tyr-Phe(NO2)-Leu-Leu. On the other hand, those of B-Phe(NO2)-Tyr-Leu-Leu for pepsin were the same or 4-20 times greater than A-Tyr-Phe(NO2)-Leu-Leu. The hydrolysis rates of the substrates by both enzymes tend to increase with the increase of the peptide chain length. The best substrate for cathepsin D was Arg-Pro-Lys-Pro-Leu-Leu-Phe(NO2)-Tyr-Leu-Leu and its kcat/Km was 1.3 μM(-1) s(-1).