Red clover necrotic mosaic virus, a member of the dianthovirus group, is characterized by a genome composed of two nonhomologous single-stranded RNAs of approximately 4.0 (RNA-1) and 1.4 kb (RNA-2). The complete nucleotide sequence of the RNA-2 has been determined. RNA-2 is 1448 nucleotides in length with a 5′ terminal m ⁷ G cap and no 3′ terminal poly-A tail or 5′ terminal VPg. An open reading frame beginning at the first initiation codon at nucleotide 80 and ending at nucleotide 1030 has been identified which can encode a polypeptide of 35 kDa. RNA-2 directs the synthesis of a 35 kDa polypeptide in vitro . SP6 and T7 transcripts from full length RNA-2 cDNA clones directed the synthesis of a polypeptide with the same electrophoretic mobility as the polypeptide directed from authentic RNA-2. Clones with various 3′ terminal deletions both outside and within the 35 kDa open reading frame were transcribed and translated in vitro to define the limits of the 35 kDa open reading frame. A second, small open reading frame capable of encoding a polypeptide of 4.9 kDa was also indicated from the sequence; however, there was no evidence for a protein product of that size. RNA-2 is presumed to be monocistronic and encode a cell-to-cell movement function. A small but significant amino acid sequence homology was observed with the brome mosaic virus RNA-3a polypeptide.