Nuclear inclusion a (NIa) protease of tobacco vein mottling virus is responsible for the processing of the viral polyprotein into functional proteins. In order to identify the active-site residues of the TVMV NIa protease, the putative active-site residues, His-46, Asp-81 and Cys-151, mere mutated individually to generate H46R, H46A, D81E, D81N, C151S, and C151A, and their mutational effects on the proteolytic activities were examined, Proteolytic activity,vas completely abolished by the mutations of H45R, H46A, D81N, and C151A, suggesting that the three residues are crucial for catalysis. The mutation of D81E decreased k(cat) marginally by about 4.7-fold and increased K-m by about 8-fold, suggesting that the aspartic acid at position 81 is important for substrate binding hut can be substituted by glutamate without any significant decrease in catalysis, The replacement of Cys-151 by Ser to mimic the catalytic triad of chymotrypsinlike serine protease resulted in the drastic decrease in k(cat) by about 1,260-fold. This result might be due to the difference of the active-site geometry between the NIa protease and chymotrypsin. The protease exhibited a bell-shaped pH-dependent profile with a maximum activity approximately at pH 8.3 and with the abrupt changes at the respective pK(a) values of approximately 6.6 and 9.2, implying the involvement of a histidine residue in catalysis. Taken together, these results demonstrate that the three residues, His-46, Asp-81, and Cys-151, play a crucial role in catalysis of the TVMV NIa protease.