We examined the role of several amino acid residues located at the N-terminus of the tobacco etch potyvirus (TEV) helper component-proteinase (HC-Pro) in virus transmissibility by aphids. Site-directed mutagenesis resulted in changes affecting amino acids that appear highly conserved among a number of potyviruses. The TEV HC-Pro amino acid residues Gly343, Val345, Ala346, Ile348, Pro355, Lys358, and Ile359 were arranged within a Cys-rich domain in a region dispensable for TEV infectivity. Two HC-Pro mutants (TEV-P355R and -K358N) exhibited a drastically reduced rate of aphid transmission whereas other mutants (TEV-G343D, -V345E, -A346H, -I348D, and -P355L) were completely unable to be aphid transmitted. In contrast, the I359M mutation had no effect on aphid transmissibility of TEV. This lack of transmissibility did not appear to be due to large differences in the amounts of both coat protein (CP) and HC-Pro in infected tobacco plants. Our results indicated that these amino acid residues likely play a highly conserved role in aphid transmission among potyviruses.