The nonstructural protein expressed from ORF-206 of turnip yellow mosaic virus is proteolytically processed to produce N-terminal 150-kDa and C-terminal 70-kDa proteins. Through the use of linker insertion and deletion analyses coupled to in vitro translation, we have delimited the protease domain to residues 731-885 encoded by the 150-kDa coding region of ORF-206. The effects of substitutions of conserved residues within this region were studied in two assays: a direct assay for proteolysis occurring during in vitro translation and an assay for viral replication in turnip protoplasts. Replication was shown to be dependent on proteolytic maturation by the failure of a mutant with an inactive protease cleavage/recognition site to amplify in protoplasts. Substitutions at Cys783 and His869 were the only ones that resulted in undetectable signals in both assays, indicating that these are probable active sites residues, most likely of a papain-like protease. Domains putatively encoding similar proteases were detected in the genomes of other tymoviruses and viruses related to tymoviruses. The putative active site cysteine residues of these domains are followed by an aliphatic amino acid, whereas an aromatic amino acid at this position is typical of cellular and previously characterized viral papain-like proteases.