The ND18 and Type strains of barley stripe mosaic hordeivirus (BSMV) differ in the local lesion phenotypes they elicit on Chenopodium amaranticolor. The ND18 strain produces large necrotic lesions on this host by 3 to 4 days postinoculation, whereas the Type strain is less virulent and elicits chlorotic local lesions which appear about 2 weeks after inoculation. We have used infectious in vitro transcripts derived from full-length cDNA clones of these two BSMV strains to investigate the genetic basis for their differential virulence on C. amaranticolor. Pseudorecombination of the wild-type alpha, beta, and gamma genomic RNAs of each strain revealed that the lesion forming phenotype segregated with RNA gamma. Fine mapping of the phenotypic determinants on RNA gamma was carried out by constructing deletion mutants, chimeric recombinants, and point mutants. These experiments showed that three different genetic elements in the Type strain RNA gamma contribute significantly to its attenuated virulence on C. amaranticolor. In addition, pseudorecombination experiments using mutant Type strain gamma RNAs that were more virulent than native Type RNA gamma indicated that the clean segregation of the lesion forming phenotype observed with wild-type RNA gamma is fortuitous. This lesion phenotype is dependent on both the multiple attenuating determinants in the wild-type Type strain RNA gamma and the source of genomic RNAs alpha and beta in the inoculum. The complexity of these virulence determinants clearly illustrates the limitations of classical pseudorecombination as a tool for the genetic analysis of plant viruses.