We have synthesized cDNA clones of the genome of the cherry strain of tomato bushy stunt virus (TBSV-cherry) and have used them as hybridization probes to identify and position two 3' coterminal subgenomic RNAs of approximately 2.2 and 0.9 kilobases (kb) in length. The 5' termini of the two subgenomic RNAs have been mapped to positions located 2156 and 936 nucleotides respectively from the 3' terminus of the viral genome. The nucleotide sequence of cDNA clones encompassing the region of the genome containing both of the subgenomic RNAs has been determined. The sequence data indicate that two nested open reading frames (ORFs) occur in the most 3' proximal location on the genome suggesting that the 0.9-kb subgenomic RNA potentially encodes two polypeptides of 19,397 and 21,610 Da. Comparison of the amino acid sequence of a potential translation product of 41,024 Da encoded by the first ORF of the 2.2-kb subgenomic RNA with the published capsid protein amino acid sequence of the BS-3 strain of TBSV indicates that the 2.2-kb subgenomic RNA encodes the capsid protein. The TBSV coat protein cistron is located internally on the genome and thus its genetic organization differs from that reported for most other small, spherical viruses with monopartite genomes. Amino acid sequence comparisons of analogous regions of the cucumber necrosis virus (CNV) genome confirms a close relationship between the viruses.