Proteins encoded by open reading frames (ORF) 3, 4, and 5 of the barley yellow dwarf luteovirus genome are translated from a single subgenomic RNA. The structural proteins are encoded by ORF 3 (coat protein) and ORF 5 (readthrough domain) and contain undefined domains that regulate the movement of virus through aphid vectors. The biological function of the nonstructural 17-kDa protein encoded by ORF 4 is unknown. A complementation method was employed to test the ability of barley yellow dwarf virions carrying mutations within the readthrough domain and the 17-kDa protein to be transmitted by aphids and to cause systemic infections in plants. We show that the readthrough domain is required for aphid transmission; however, it is not required for virus to be taken up by aphid hindgut cells and released into the hemocoel. The circulative pathway of luteoviruses in aphid vectors requires that virus be actively transported from the hemolymph into the salivary system. Thus, it appears that the readthrough domain is required for transport of virus through membranes of the aphid salivary glands. Furthermore, the readthrough domain was not required for systemic infection of plants, but did influence the accumulation of virus in infected plants. The 17-kDa protein is required for the systemic infection of plants.