Turnip mosaic virus (TuMV) NIa protease cleaves the viral polyprotein at seven distinct junctions out of nine. The amino acid sequences of the seven cleavage sites have three conserved amino acids, V, H, Q in positions P4, P2, P1, respectively. Small molecules as well as conjugated peptides were tested for proteolytic activity of the enzyme. None of small molecules tested, such as methylumbelliferyl-p-guanidinobenzoate, p-nitrophenyl-p'-guanidinobenzoate, p-nitrophenyl acetate, and methylumbelliferyl-N-acetylglutamate, were hydrolyzed. Ac-V-Y-H-Q-Mca was also not hydrolyzed. Intramolecularly quenched fluorogenic substrates Dns-P-V-Y-H-Q-A-W-NH(2) and Dns-P-V-Y-H-Q-W-NH(2) emitted fluorescence after addition of TuMV NIa protease. The proteolysis rate of Dns-P-V-Y-H-Q-A-W-NH(2) was comparable to that of the tetradecapeptide with an optimum sequence, but Dns-P-V-Y-H-Q-W-NH(2) was hydrolyzed at a slower rate, which was confirmed independently by HPLC analysis. These results suggest that intramolecularly quenched fluorogenic substrates can be used for the continuous assay of TuMV NIa protease.