Highly infectious synthetic transcripts from full-length cucumber necrosis virus (CNV) cDNA clones have been prepared. Infections produced by these transcripts, by CNV RNA, and by mutated transcripts are compared. Inoculation with natural CNV RNA resulted in a mild systemic necrosis in Nicotiana clevelandii, whereas inoculation with synthetic CNV transcripts resulted in severe systemic necrosis. An abundant low molecular weight RNA species was associated with CNV RNA infections but was not detected in synthetic transcript infections. Differences in the symptoms and the RNA profiles produced by these two inoculums indicate that our laboratory strain of CNV contains defective interfering RNAs. Studies using mutant synthetic CNV transcripts which contain point substitutions in the AUG codons for two 3' terminal nested open reading frames encoding 21- and 20-kDa proteins are also described. Evidence is presented which suggests that both the 21- and 20-kDa proteins are produced during CNV infection, although only one subgenomic mRNA which can encode both of these proteins can be detected in infected plants. Together, these results indicate that the CNV 21- and 20-kDa proteins are expressed from a bifunctional subgenomic mRNA generated during CNV infection.