The first approximately 60 amino acids of the N-terminal part of the potyvirus helper component-proteinase (HC-Pro) include highly conserved residues comprising a Cys-rich region. In the present study, the domain in Potato virus Y sufficient for self-interaction was mapped using the yeast two-hybrid system to the 83 N-terminal amino acids of HC-Pro. Mutations in the conserved His and two Cys residues within the Cys-rich region have a strong debilitating effect on self-interaction when introduced in the full-length HC-Pro, but not when introduced in the N-terminal fragment.