In vitro transcripts from mixtures of appropriate brome mosaic virus (BMV) cDNA clones are infectious when inoculated onto barley plants. Infectivity depends on in vitro transcription and on the presence of transcripts from clones of all three BMV genetic components. Infectivity is destroyed by RNase after transcription, but it is insensitive to RNase before or to DNase after transcription. Virion RNAs from plants infected with cDNA transcripts hybridize to BMV-specific probes and coelectrophorese with virion RNAs propagated from conventional inoculum. Direct RNA sequencing shows that a deletion in the noncoding region of one infectious BMV clone is preserved in viral RNA from plants systemically infected with transcript mixtures representing that clone.