RNA-dependent RNA polymerases (RdRp) isolated from bamboo mosaic potexvirus (BaMV) and potato virus X infected Nicotiana benthamiana plants and solubilized with the detergent NP-40, generated a full-length genomic and two subgenomic double-stranded RNAs of respective viruses in an in vitro RdRp assay containing endogenous RNA templates. Template-dependent and species-specific RdRp activity could be detected after the removal of endogenous RNA templates. The 3' untranslated regions (UTR) containing a stretch of 40 adenylate residues were shown to be an efficient exogenous RNA template for in vitro RdRp reactions. Solution hybridization and nuclease digestion studies revealed that the products transcribed in vitro were minus-sense. Besides using the 3' UTR for minus-sense RNA synthesis, the BaMV RdRp can also recognize 3' terminal 77 nucleotides of the minus-strand for plus-sense RNA synthesis. Promoter studies with BaMV RdRp showed that domain D containing the potexviral hexamer motif of the 3' UTR would be the major contributor of minus-sense RNA synthesis in vitro. On the other hand, the pseudoknot domain containing the poly(A) sequences would be sufficient for minus-sense RNA synthesis.