Fluorogenic peptide substrates with fluorophore/ quencher-capped ends have found extensive use in monitoring protease activity in the screening of small-molecule libraries for protease inhibitors. We report here the identification and characterization of a fluorogenic substrate for tumor necrosis factor-␣ converting enzyme (TACE). This substrate is a 10-amino-acid peptide (LAQAVRSSSR) capped with an o-aminobenzoyl group on the N-terminal end and with a 3-(2,4-dinitrophenyl)-L-2,3-diaminopropionic amide group on the C-terminal end. Exhaustive enzymatic conversion of the substrate to products resulted in a fluorescence enhancement of ϳ11-fold. A single cleavage occurred at the A-V scissile bond of the peptide. The validity of this fluorimetric assay for TACE was corroborated by an independent HPLC method. Interestingly, the hydrolysis of the substrate displayed positive cooperativity with a Hill coefficient of 1.5, while the hydrolysis of the corresponding uncapped peptide displayed Michaelis-Menten kinetics. A k cat value of 21.6 s ؊1 and an S 0.5 value of 342 M were obtained for the fluorogenic substrate. The addition of the two capping groups on the two ends of the peptide enhanced the k cat value by 64-fold. Nine additional decapeptides that contained the same capping groups on the two ends and substitutions at the P1 and P1 sites were also tested. TACE appears to slightly prefer the A-V scissile bond. The enzyme also cleaves scissile bonds such as F-V, A-I, and A-L efficiently.