Replication by many positive-strand RNA viruses includes genomic RNA amplification and subgenomic mRNA (sgRNA) transcription. For brome mosaic virus (BMV), both processes occur in virus-induced, membrane-associated compartments, require BMV replication factors 1a and 2a, and use negative-strand RNA3 as a template for genomic RNA3 and sgRNA syntheses. To begin elucidating their relations, we examined the interaction of RNA3 replication and sgRNA transcription in Saccharomyces cerevisiae expressing 1a and 2a, which support the full RNA3 replication cycle. Blocking sgRNA transcription stimulated RNA3 replication by up to 350%, implying that sgRNA transcription inhibits RNA3 replication. Such inhibition was independent of the sgRNA-encoded coat protein and operated in cis. We further found that sgRNA transcription inhibited RNA3 replication at a step or steps after negative-strand RNA3 synthesis, implying competition with positive-strand RNA3 synthesis for negative-strand RNA3 templates, viral replication factors, or common host components. Consistent with this, sgRNA transcription was stimulated by up to 400% when mutations inhibiting positive-strand RNA3 synthesis were introduced into the RNA3 5′-untranslated region. Thus, BMV subgenomic and genomic RNA syntheses mutually interfered with each other, apparently by competition for one or more common factors. In plant protoplasts replicating all three BMV genomic RNAs, mutations blocking sgRNA transcription often had lesser effects on RNA3 accumulation, possibly because RNA3 also competed with RNA1 and RNA2 replication templates and because any increase in RNA3 replication at the expense of RNA1 and RNA2 would be self-limited by decreased 1a and 2a expression from RNA1 and RNA2.