Purified particles of barley yellow dwarf luteovirus (BYDV) contain a major 22-kDa protein and a minor protein of approximately 58 kDa. The 22-kDa capsid protein is encoded by open reading frame (ORF) 3. ORF 5 is immediately downstream and in frame with ORF 3 and a 72-kDa protein can be translated via a readthrough suppression of the ORF 3 termination codon. Antibodies were produced against two Escherichia coli expressed polypeptides that represent the amino- and carboxyl-terminal halves of a putative 50-kDa protein encoded by ORF 5. Immunological analyses indicated that the 58-kDa protein associated with purified virions contained sequences encoded by ORF 3 and ORF 5. The carboxyl terminal portion of the full-length (72 kDa) readthrough protein was absent from the 58-kDa protein. The full-length readthrough protein was detected in infected oat protoplasts and plant tissue, but was not associated with virus particles purified from plants. The carboxyl-terminal portion of the 72-kDa readthrough protein was not required for aphid transmission; however, virus was transmitted more efficiently from protoplast extracts containing virions and soluble 72-kDa readthrough protein than from mock-inoculated protoplast extracts to which plant purified virus was added. The full-length readthrough protein, although not required for transmission, may increase the transmission efficiency of BYDV by aphids.