Tobacco Etch Virus Protease (TEV protease) is widely used as a tool for separation of recombinant target proteins from their fusion partners. The crystal structures of two mutants of TEV protease, the active autolysis-resistant mutant TEV-S219D in complex with the proteolysis product, and the inactive mutant TEV-C151A in complex with a substrate, have been determined at 1.8 and 2.2 Å resolution, respectively. The active sites of both mutants, including their oxyanion holes, have identical structures. The C-terminal residues 217–221 of the enzyme are involved in formation of the binding pockets S 3–S 6. This indicates that the autolysis of the peptide bond Met218–Ser219 exerts a strong effect on the fine-tuning of the substrate in the enzyme active site, which results in a considerable decrease in the enzymatic activity.