The barley yellow dwarf virus (BYDV) coat protein gene is separated from an adjacent downstream open reading frame (ORF) by a single termination codon. Immunological analysis of this downstream "readthrough" region reveals multiple coat protein-readthrough products. A full-length 72-kDa (P72) coat protein-readthrough fusion product is detected in total lysates from infected cells. However, purified aphid transmissible virions contain only a 50-kDa (P50) coat protein-readthrough product. Virion-associated P50 lacks the C-terminal domain predicted by its ORF sequence. A separate 33-kDa polypeptide (P33) corresponding to the readthrough C-terminus domain is detected in the crude cellular membrane fraction. Site-directed and deletion mutational analysis demonstrate that the readthrough ORF is dispensable for BYDV replication and virion accumulation in protoplasts. In contrast, a mutant which results in a continuous fusion product of coat and readthrough sequences is not viable. Point mutations were used to map regions required for P50 and P72 synthesis. A model explaining the relationships between the three forms of the readthrough polypeptides is proposed.