A continuous fluorimetric assay for tail-specific protease (Tsp) has been developed using a fluorescence donor/quencher system, in which 5-[(2-aminoethyl) amino]naphthalene-1-sulfonic acid (EDANS) and 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) are attached to the N-terminus and the lysyl side chain of peptide AARAAK-(6-aminocaproyl) 2 -ENYALAA, respectively. Tsp-mediated cleavage of the Ala-Arg peptide bond separates the quencher, DABCYL, from the donor, EDANS, and results in a large increase in the fluorescent yield of EDANS (>50-fold). Using this sensitive assay,Escherichia colitail-specific protease was shown to exhibit typical Michaelis-Menten kinetics with ak c a t of 0.086 +/- 0.002 s - 1 ,K M of 4.0 +/- 0.3 μM, andk c a t /K M of 2.2 x 10 4 M - 1 s - 1 . A control substrate, which only differs from the above substrate by having a charged residue (glutamate) at the C-terminus, showed drastically reduced activity to Tsp (k c a t /K M = 58 M - 1 s - 1 ). A peptide containing the C-terminal sequence of the substrate, GRGYALAA, was shown to be a competitive inhibitor of Tsp with aK I value of 31 μM. These results demonstrate the utility of this assay for the rapid assessment of Tsp activity.