In order to understand the relationship between replication and aminoacylation of the genomic RNAs of brome mosaic virus, the replication of four mutants, whose RNAs were expected (on the basis of their properties in vitro) to be inefficiently tyrosylated in vivo, was studied in barley protoplasts and plants. Test inocula consisted of capped transcripts of wild-type RNAs 1 and 2, and of RNA 3 variants with defined mutations in the 3' tRNA-like region. Mutant 5'PsK, which is defective in minus-strand promoter activity and a poor substrate in vitro for both tyrosylation and 3' adenylation, replicated in protoplasts to 20% of wild-type even though only about 6% of the progeny molecules had correct 3' termini that would permit tyrosylation. Mutant psi GG, which is defective in vitro for 3' adenylation and minus-strand promoter activities but accepts tyrosine at near-normal rates, replicated to 40% of wild-type in protoplasts although only 15% of the progeny molecules had correct 3' termini. Two other mutants (delta 5' and 5'AGA), with 20-fold lower rates of tyrosylation in vitro than wild-type RNA, replicated to 60 to 70% of wild-type levels in protoplasts and gave similar yields to wild-type in systemic infections of plants. All mutant sequences were preserved in progeny RNAs, indicating that no recombination between homologous 3' ends occurred. The 40% reduction of replication in protoplasts seen for mutant delta 5', whose only known functional lesion is depressed tyrosylation in vitro, may indicate that an indirect role for aminoacylation exists. However, the results obtained argue against an obligatory role for tyrosylation in RNA replication in vivo.