Via a combination of chemical and enzymatic synthesis, new hexapeptide substrates convenient for use in activity assessment of several aspartyl proteinases--porcine pepsin, human pepsin, gastricsin, and cathepsin D--were prepared. These peptide derivatives, o-aminobenzoyl-Ala-Ala-Phe-Phe-Ala-Ala-p-nitroanilide and N-(o-aminobenzoyl-Ala-Ala-Phe-Phe-Ala-Ala)-N'-2,4-dinitrophenyl ethylenediamine, contain a fluorescent o-aminobenzoyl moiety as well as p-nitroaniline or N-2,4-dinitrophenyl ethylenediamine--the groups that cause fluorescence quenching. Aspartyl proteinases hydrolyze the Phe-Phe peptide bond in the substrates, which diminishes quenching due to separation of the fluorescent and quenching moieties and leads to an increase in the fluorescence intensity of o-aminobenzoyl residue. Abz-Ala-Ala-Phe-Phe-Ala-Ala-Ded, being fairly well hydrolyzed by HIV proteinase, might be used for assay of this enzyme.