Barley stripe mosaic virus (BSMV), the type member of the hordeivirus group, has a plus-stranded genome comprising RNA species designated alpha, beta, and gamma. Although RNA beta is essential for infection of whole plants, it is dispensable for infection of barley protoplasts. We have used a full-length cDNA clone of RNA beta from which infectious in vitro transcripts can be derived to construct a number of mutations in its four genes. Mutations introduced into the beta b, beta c, or beta d genes eliminated infectivity of the RNA. The coat protein and the RNA sequences encoding the coat protein were completely dispensable for infection of barley plants by BSMV, and no detrimental effect on systemic movement of the virus was observed. However, besides eliminating coat protein expression in vivo, mutations within the coat protein gene and the first intercistronic region affected a number of other phenotypes: (1) expression of a downstream gene (beta b), (2) stability of the genomic RNA during virus multiplication in planta, (3) the requirement for a trans-acting BSMV protein (gamma b), (4) symptomatology and disease development in infected barley plants, and (5) host range.