Coinfections of synthetic transcripts from a cDNA clone of cucumber necrosis virus (CNV) and cDNA clones of defective interfering (DI) RNAs were previously demonstrated to contain DI-like RNAs approximately twice the size of the DI RNA used for co-inoculation. Analysis of these RNAs revealed that they are head-to-tail repeats of CNV DI RNA sequence (dimers). Sequence analysis of 21 cloned dimer junctions indicated that approximately half of the junction sequences correspond to precise fusions of monomer units. A cDNA clone corresponding to a dimer of DI RNA 42 was constructed. Synthetic DI RNA 42 dimer transcripts were biologically active in coinfections, resulting in the accumulation of high levels of DI RNA 42 monomers. The possibility that dimers serve as templates for the generation of DI RNA monomers is discussed.