The nucleotide sequences of RNA-5 from two laboratory isolates (D-5 and D-6) and three field isolates (SH1, S43 and R83) of beet necrotic yellow vein virus (BNYVV) were determined. Isolates D-5 and D-6, derived from a D field culture during mechanical inoculation, contained RNA-5 of molecular size 1.4 kb and 1.0 kb, respectively. The sequences of D-5 SH1, S43 and R83 were found to be at least 98% identical and from 1342 to 1347 nucleotides in length, excluding the poly(A) tail. Each contained a single open reading frame (ORF) encoding a 228 amino acid protein with a molecular mass of 26189 Da (P26). The coding sequence was bordered by a long leader of 443 to 448 nucleotides and a 3'-terminal non-coding region of 215 nucleotides. In isolate D-6, containing the smaller approximately 1.0 kb RNA species referred to as RNA-5a, the ORF had undergone an internal deletion of 303 nucleotides. No sequence identity was found between RNA-5 and either RNA-3 or RNA-4, except for the 5'-terminal nine residues and for approximately the 3'-terminal 200 residues. Thus, the genome organization of BNYVV RNA-5 is very similar to that of RNA-3 and RNA-4, both of which are essential for survival of BNYVV in nature. Although RNA-5 is not essential, it may be associated with symptom expression of BNYVV.