The helper component-proteinase (HC-Pro) encoded by potyviruses functions to cleave the viral polyprotein by an autoproteolytic mechanism at the HC-Pro C-terminus. This protein belongs to a group of viral cysteine-type proteinases and has been shown previously to catalyze proteolysis between a Gly-Gly dipeptide. The amino acid sequence requirements surrounding the HC-Pro C-terminal cleavage site of the tobacco etch virus polyprotein have been investigated using site-directed mutagenesis and in vitro expression systems. A total of 51 polyprotein derivatives, each differing by the substitution of a single amino acid between the P5 and P2' positions, were tested for autoproteolytic activity. Substitutions of Tyr (P4), Val (P2), Gly (P1), and Gly (P1') were found to eliminate or nearly eliminate proteolysis. Substitutions of Thr (P5), Asn (P3), and Met (P2'), on the other hand, were permissive for proteolysis, although the apparent processing rates of some polyproteins containing these alterations were reduced. These results suggest that auto-recognition by HC-Pro involves the interaction of the enzymatic binding site with four amino acids surrounding the cleavage site. Comparison of the homologous sequences of five potyviral polyproteins revealed that the residues essential for processing are strictly conserved, whereas the nonessential residues are divergent. The relationship between HC-Pro and other viral and cellular cysteine-type proteinases is discussed.