An in vitro system was applied to analyze the replication of a satellite RNA of Bamboo mosaic virus (BaMV), designated satBaMV RNA, using solubilized membrane-bound RNA-dependent RNA polymerase (RdRp) complexes isolated from BaMV-infected Nicotiana benthamiana. After removal of endogenous templates, the RdRp complexes of BaMV catalyzed RNA synthesis upon the addition of the full-length positive (+)- or negative (-)-strand satBaMV RNA transcripts used as templates. Both (+)- and (-)-satBaMV RNA products were detected when only the (+)-satBaMV RNA was used as a template in the in vitro RdRp assays, which further demonstrated the capability of the RdRp preparation to complete the replication cycles of satBaMV RNAs. In addition, use of 5' rapid amplification of cDNA ends and DNA sequencing showed that the BaMV RdRp preparation could specifically recognize the promoter sequences in the (-)-satBaMV RNA for accurate initiation of (+)-satBaMV RNA synthesis. The results suggested that the same enzyme complexes could be used for the replication of both BaMV genomic and satBaMV RNAs. The soluble and template-dependent RdRp could be further used in mechanistic studies, such as those analyzing the cis-elements and candidate host factors required for satBaMV RNA replication in vitro.