The RNA genome of tobacco etch virus (TEV) encodes a large polyprotein precursor that is processed to mature proteins by virus‐specific proteinases. Cleavage sites located within the carboxyl‐terminal two‐thirds of the polyprotein are processed by a TEV‐encoded 49 kd proteinase, while the enzyme(s) responsible for cleaving the remaining sites has not been found. In this study, a second TEV‐encoded proteinase has been identified based on cell‐free expression of defined RNA transcripts. The boundaries of this proteinase have been delineated by deletion analysis and site‐directed mutagenesis. The proteolytically active domain has been localized to the carboxyl‐terminal half of the 56 kd aphid‐transmission helper component. A cleavage site that is recognized by this proteinase has been identified in the polyprotein adjacent to the carboxyl‐terminus of the enzyme, and the proteinase appears to cleave by an autocatalytic mechanism. Proteolysis in vitro occurs between a Gly‐Gly dipeptide as determined by radiochemical sequencing at the amino‐terminus of the proteolytic product.