Rubella virus (RV) genomic RNA contains two large open reading frames (ORFs): a 5'-proximal ORF encoding nonstructural proteins (NSPs) that function primarily in viral RNA replication and a 3'-proximal ORF encoding the viral structural proteins. Proteolytic processing of the RV NSP ORF translation product p200 is essential for viral replication. Processing of p200 to two mature products (p150 and p90) in the order NH₂-p150-p90-COOH is carried out by an RV-encoded protease residing in the C-terminal region of p150. The RV nonstructural protease (NS-pro) belongs to a viral papain-like protease family that cleaves the polyprotein both in trans and in cis. A conserved X domain of unknown function was found from previous sequence analysis to be associated with NS-pro. To define the domains responsible for cis- and trans-cleavage activities and the function of the X domain in terms of protease activity, an in vitro translation system was employed. We demonstrated that the NSP region from residue 920 to 1296 is necessary for trans-cleavage activity. The domain from residue 920 to 1020 is not required for cis-cleavage activity. The X domain located between residues 834 and 940, outside the regions responsible for both cis- and trans-cleavage activities of NS-pro, was found to be important for NS-pro trans-cleavage activity but not for cis-cleavage activity. Analysis of sequence homology and secondary structure of the RV NS-pro catalytic region reveals a folding structure similar to that of papain.