A full-length cDNA of cucurbit aphid-borne yellows virus (CABYV) has been constructed and expressed either as anin vitrotranscript, under control of a bacteriophage T7 RNA polymerase promoter, orin vivo,under control of the cauliflower mosaic virus 35S promoter in an agroinfection vector. The biological activity of the cloned cDNA was demonstrated by the ability of itsin vitrotranscript to replicate in protoplasts and of the agroinfection vector to infect agroinoculated plants. Virus in the agroinfected plants could be transmitted by the aphid vectorsMyzus persicaeandAphis gossypii.The specificity of luteovirus RNA packaging was investigated by replacing (1) the CABYV coat protein gene (and the overlapping ORF5) by the corresponding region of potato leafroll luteovirus or (2) the CABYV readthrough domain by the readthrough domain of beet western yellows luteovirus. The resulting chimeric transcripts replicated in protoplasts and produced virions.