Abstract RNA extracted from purified barley stripe mosaic virus (BSMV) was analyzed by density gradient centrifugation and polyacrylamide gel electrophoresis. All strains tested had a prominent 21.3 S RNA, which was separated into two components by gel electrophoresis. Most preparations of North Dakota 18 and Argentina Mild strains had an additional 19.5 S component, which was resolved into one or two RNA species by gel electrophoresis. Some preparations also contained variable amounts of two or three minor RNA components migrating slowly in gels; these components sedimented at about 30 S in sucrose gradients. The two 19.5 S components were more variable than other components, and occasionally one or both disappeared during virus subculture. Double-stranded RNA isolated from infected plants migrated more slowly in polyacrylamide gels than did single-stranded RNA isolated from purified virions, but both types of RNA from the same strain had the same number of major RNA components. The RNAs of BSMV migrated more slowly in gels than expected from their sedimentation rates if brome mosaic virus RNAs and barley ribosomal RNAs were used as standards, which suggests that BSMV RNAs and the standard RNAs differ in secondary configuration. Mixtures of the separated RNA components were more infectious than single components, which indicates that more than one RNA component was necessary to establish infection.