HIV-1 protease hydrolyzed the newly synthesized peptides, Asn-Asn-Gln-Val-Phe (NO₂)-Val-Arg-NH₂ and acetyl-Arg-Lys-Leu-Val-Phe(NO₂)-Leu-Asp-Gly-NH₂ between the valyl and (p-nitro)phenylalanyl residues. The hydrolysis of these peptides resulted in a decrease in UV absorbance at 310 nm. The HIV-1 protease-catalyzed peptidolysis of these two peptide substrates was characterized by a linear time course at substrate turnover of ≤20%. The solubilities of these substrates at pH 4.7 were sufficient to perform initial rate measurements over a concentration range of 50 to 500 nM. Steady-state kinetic data and inhibition constants of the peptidolysis of these peptide substrates resulted in comparable values.