The nuclear inclusion protein a (NIa) of turnip mosaic potyvirus is a protease processing the viral polyprotein into functional proteins. It has been shown that the NIa C-terminal 27-kDa protease cleaves itself between Ser-223 and Gly-224 to generate a 25-kDa protein lacking the C-terminal 20 amino acids. We have found a second internal cleavage near the C-terminus resulting in the degradation of the 25-kDa protein into a 24-kDa protein. Substitution of the active site Asp-81 or Cys-151 with Asn or Ser, respectively, prevented the second cleavage, suggesting that the internal cleavage is also due to the proteolytic activity of the NIa protease. This second internal cleavage was found to occur between Thr-207 and Ser-208, eliminating the C-terminal 36 amino acids from the 27-kDa protease. The proteolytic activity of the 24-kDa protein was not detected at all when it was measured using a nonapeptide containing the cleavage site between 6K1 and CI as a substrate, suggesting that the C-terminal region between residues 208 and 223 contains essential amino acids for the processing of 6K1-CI polyprotein. The deletion analyses of the C-terminal region revealed that at least 217 amino acids from the N-terminus are required for the catalytic activity of the NIa protease. The point mutation of Trp-212 to Ser, Gly-213 to Ser, or Ile-217 to Asp drastically abolished the catalytic activity, demonstrating that Trp-212, Gly-213, and Ile-217 are important for the processing of 6K1-CI polyprotein.