The 28-kDa protease from human cytomegalovirus (hCMV) has been successfully cloned, expressed, and purified to homogeneity. An internally quenched fluorescent substrate (4-4'-dimethylaminophenazo)benzoyl-Arg-Gly-Val-Val-Asn-Ala-Ser-Ser -Arg-Leu-Ala-5-[(2'-aminoethyl)-amino]-naphthalene-1-sulfonic acid; DABCYL-CMV-EDANS) based on the maturational cleavage site (M-site) junction was synthesized in an effort to develop a fluorescence-based assay. This substrate is cleaved specifically between the Ala-Ser peptide bond thereby liberating the C-terminal peptide-EDANS fragment from the proximity quenching effect of the DABCYL group. This results in greater than a 10-fold increase in fluorescence that is observed at the EDANS emission wavelength of 495 nm. Human CMV protease efficiently cleaved this synthetic substrate permitting continuous assay at peptide concentrations lower than 10 microM. At substrate concentrations greater than 10 microM, linearity was lost due to the "inner filter effect." This represents the first fluorescence-based assay for any of the herpes virus proteases. Additionally, a peptidyl inhibitor, H-Arg-Gly-Val-Val-Asn-Ala-psi[CH2NH]-Ser-Ser-Arg-Leu-Ala-OH, was prepared. This inhibitor was also based on the same M-site cleavage junction with a nonhydrolyzable reduced peptide bond incorporated at the cleavage site. Using the fluorescence-based assay, this reduced peptide bond analog was observed to be an inhibitor of hCMV protease with an inhibition constant of > 500 microM.