Barley stripe mosaic virus (BSMV) has a tripartite genome comprising RNAs designated alpha, beta, and gamma, which collectively encode seven polypeptides. We show here that an antiserum raised against an abundant disease-specific protein from BSMV-infected plants reacts specifically with the viral beta b gene product expressed as part of a beta-galactosidase fusion protein in Escherichia coli. Two predominant forms of the protein, beta b and beta b', are synthesized in vivo. Infectious in vitro transcripts derived from wild-type and mutant BSMV cDNA clones have been used to map the initiation site for translation of the beta b protein in vivo. The results of our mutagenesis experiments are consistent with a model in which translation of the beta b' protein is initiated by ribosomes that scan past the 5'-proximal beta b initiation site. A mutant which is able to synthesize only the shorter beta b' protein was indistinguishable from the wild-type with respect to all of the phenotypes tested. Thus, the beta b form of the protein is dispensable in planta, and whether the two forms of the protein have different functions in vivo is unclear at present.