The central portion of the brome mosaic virus (BMV) 2a protein represents the most conserved element among the related RNA replication components of a large group of positive-strand RNA viruses of humans, animals, and plants. To characterize the functions of the 2a protein, mutations were targeted to a conserved portion of the 2a gene, resulting in substitutions between amino acids 451 and 484. After the temperature profile of wild-type BMV RNA replication was defined, RNA replication by nine selected mutants was tested in barley protoplasts at permissive (24 degrees C) and nonpermissive (34 degrees C) temperatures. Four mutants did not direct RNA synthesis at either temperature. Various levels of temperature-sensitive (ts) replication occurred in the remaining five mutants. For two ts mutants, no viral RNA synthesis was detected at 34 degrees C, while for two others, an equivalent reduction in positive- and negative-strand RNA accumulation was observed. For one mutant, positive-strand accumulation was preferentially reduced over negative-strand accumulation at 34 degrees C. Moreover, this mutant and another displayed preferential suppression of genomic over subgenomic RNA accumulation at both 24 and 34 degrees C. The combination of phenotypes observed suggests that the 2a protein may play a role in the differential initiation of specific classes of viral RNA in addition to a previously suggested role in RNA elongation.