Barley stripe mosaic Hordeivirus (BSMV) is a positive-strand RNA virus requiring three single-stranded RNAs (alpha, beta, and gamma) for infectivity. A terminal-sequence-dependent cloning strategy was used to clone the entire genome of the CV17 strain. Full-length gamma cDNA clones were obtained when oligonucleotides specific for the 5'-terminal sequence of RNA alpha were used in the cloning procedure, but not when RNA gamma-specific oligonucleotides were used. Sequence analysis of six putative gamma cDNA clones revealed that nucleotides 1-70 possess 89% homology with the first 70 nucleotides of RNA alpha. This leader region is separated from the gamma-specific coding region by an eight-base intervening sequence common to both CV17 RNAs alpha and gamma. Northern and Southern hybridization with oligonucleotide probes specific for either alpha or gamma leader sequences indicated that CV17 gamma cDNA clones are representative of native CV17 gamma RNAs. Furthermore, bioassays indicated that in vitro transcripts derived from these gamma cDNA clones were infectious when coinoculated with in vitro transcripts of full-length alpha and beta cDNA clones. Thus, the evidence suggests that RNA gamma of BSMV strain CV17 is a recombinant molecule which may have arisen as a result of natural recombination between RNAs alpha and gamma.