Transfection of barley protoplasts with brome mosaic virus (BMV) RNAs 1 + 2 in the absence of RNA-3 yielded a molar ratio for (+):(-)-strand progeny at 24 hr postinoculation near unity, whereas over 100-fold more (+)- than (-)-strand progeny accumulated in its presence. The presence of RNA-3 enhanced total (+)-strand RNA production 205-fold and that of RNAs 1 + 2 by 29-fold. In contrast, total (-)-strand RNA accumulation decreased by 68% and that for (-)RNAs 1 + 2 by 79% in the presence of RNA-3. Transfections containing an RNA-3 mutant (Gsgi----U RNA-3) that is incapable of yielding RNA-4 as a result of a single nucleotide substitution at the subgenomic RNA initiation site yielded only 66% of the (+):(-) asymmetry seen in the presence of wild-type RNA-3. Only 1.8-fold excess of (+)-over (-)-strand production was obtained for transfections that included delta SGP RNA-3, a deletion that includes the subgenomic promoter core and extends 43 nt into the RNA-4 sequence. Transfections containing RNA-3 mutants bearing frameshifts or deletions in the coat protein cistron yielded levels of asymmetry similar to those seen for Gsgi----U RNA-3. These findings implicate the subgenomic promoter and other sequences in the intercistronic region of RNA-3 as the primary determinants of asymmetric replication, although the coat protein may be an additional factor enhancing the accumulation of (+)-strand RNA.