Two virus-encoded proteinases are responsible for proteolysis of potyvirus polyproteins. One of these, HC-Pro, is a multifunctional protein that autolytically cleaves at its carboxyl-terminus (J.C. Carrington et al., 1989, EMBO J. 8, 365-370). To identify the class of proteinase to which HC-Pro belongs, tobacco etch virus (TEV) HC-Pro mutants containing single amino acid substitutions at serine, cysteine, aspartic acid, and histidine positions were synthesized by in vitro transcription and translation and were tested for autoproteolytic activity. Combinations of these residues are constituents of the active sites of diverse groups of cellular and viral proteinases. Only those positions that were strictly conserved among four potyvirus HC-Pro proteolytic domains (for which sequences have been deduced) were mutagenized. Of the 19 mutant proteinases synthesized and tested, only those with alterations at Cys-649 and His-722 were defective for HC-Pro autolytic activity. Most of the other mutant proteinases exhibited no impairments in processing kinetics experiments. The spectrum of essential residues, as defined by this genetic analysis, supports the hypothesis that HC-Pro most closely resembles members of the cysteine-type family of proteinases.