The 11-kD protease (PR) encoded by the human immunodeficiency virus 1 (HIV-1) is essential for the correct processing of viral polyproteins and the maturation of infectious virus, and is therefore a target for the design of selective acquired immunodeficiency syndrome (AIDS) therapeutics. To facilitate the identification of novel inhibitors of HIV-1 PR, as well as to permit detailed studies on the enzymology and inhibition of this enzyme, a continuous assay for its activity was developed that was based on intramolecular fluorescence resonance energy transfer (RET). The assay used the quenched fluorogenic substrate 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL)--Ser Gln Asn Tyr Pro Ile Val Gln--5-[(2-aminoethyl)amino]naphthalene-1 sulfonic acid (EDANS), whose peptide sequence is derived from a natural processing site for HIV-1 PR. Incubation of recombinant HIV-1 PR with the fluorogenic substrate resulted in specific cleavage at the Tyr-Pro bond and a time-dependent increase in fluorescence intensity that was linearly related to the extent of substrate hydrolysis. An internally quenched fluorogenic substrate was also designed that was selectively cleaved by the related PR from avian myeloblastosis virus (AMV). The fluorescence quantum yields of the HIV-1 PR and AMV PR substrates in the RET assay increased by 40.0- and 34.4-fold, respectively, per mole of substrate cleaved. Because of its simplicity, rapidity, and precision in the determination of reaction rates required for kinetic analysis, this method offers many advantages over the commonly used high-performance liquid chromatography- or electrophoresis-based assays for peptide substrate hydrolysis by retroviral PRs.