RNA3 of the Kin strain of the tripartite (+)-strand cucumber mosaic virus has 2199 nucleotide residues. Two open reading frames encoding 3a protein (a putative movement protein) and coat protein (CP) are separated by a 286-nucleotide inter-cistronic region (IR). This IR contains a subgenomic promoter for production of a subgenomic RNA (RNA4), from which CP is synthesized. Using transcripts generated from mutant forms of a cDNA clone of RNA3, we have characterized the cis-acting sequences necessary for RNA3 accumulation and RNA4 synthesis and analyzed the role of the RNA3-encoded proteins. Efficient accumulation of RNA3 derivatives in tobacco protoplasts required 92 nucleotides at the 5' end, 250 nucleotides in the IR, and 275 nucleotides at the 3' end of the RNA molecule. The 250-nucleotide IR includes a 90-nucleotide sequence which is necessary for subgenomic promoter activity. Although common regions are involved in RNA3 accumulation and RNA4 synthesis, the modes of action of IR for these two phenomena are different. The analysis of forms of RNA3 with internal duplications demonstrated that RNA3 accumulation depended on the context of the IR. Subgenomic promoter activity was more position dependent and was always stronger from the promoter closer to the 3' end of the (+)-strand RNA. A mutation in IR specifically affected (+)-strand RNA accumulation, indicating a role for that region in (+)-strand synthesis. The role of the RNA3-encoded proteins was analyzed by mutation and inoculation either to plants or to protoplasts. Mutation of the 3a protein had no effect on RNA3 accumulation in protoplasts, whereas CP mutations caused reduced CMV RNA accumulation. This reduction was more pronounced for (+)- than for (-)-strand accumulation. RNA of CP mutations was undetectable in inoculated leaves, whereas RNA of 3a protein mutants accumulated, albeit at levels several orders of magnitude lower than with wild-type CMV. The conclusion from these data is that both proteins are required for efficient spread of CMV from the site of infection.