Abstract The nature of the polypeptide products encoded by the 5′-terminal region of the RNA of the potyvirus, tobacco vein mottling virus (TVMV), was investigated. Single-stranded DNA probes complementary to either nucleotides 1100–2100 or 2100–2820 from the 5′ terminus of the RNA were prepared by subcloning recombinant plasmids in bacteriophage M13. These were hybridized to TVMV RNA, the DNA:RNA hybrids translated in a reticulocyte lysate cell-free translation system (hybrid-arrested translation), and the products analyzed by electrophoresis on SDS-containing polyacrylamide gels. The hybrids produced altered patterns of polypeptides which indicated that the major product, P75, was translated from the 5′ terminus of the RNA. The N-terminal portion (molecular weight approximately 35,000) of P75 did not react with antisera to any of the five known potyviral proteins, suggesting the existence of a previously unidentified cistron at the 5′ terminus. Translation of additional RNA sequences produced a polypeptide with a molecular weight of 68,000 which was immunoprecipitable by antiserum to TVMV helper component, establishing the coding region for helper component near the 5′ terminus. Limited DNA sequence analysis of the region encoding the C terminus of P75 revealed an open reading frame of 369 nucleotides followed by a pair of UAG Codons. Pulse-chase experiments demonstrated that in the first 10 min of incubation, the only polypeptide initiated was P75, suggesting that products downstream from P75 arose from in vitro fragmentation of TVMV RNA and translation of the RNA fragments.