RNA-dependent RNA polymerase activity was detected in a membrane-free cytoplasmic fraction from the leaves of Nicotiana clevelandii 4 days after inoculation with velvet tobacco mottle virus (VTMoV). The amount of enzyme activity was still increasing 8 days after inoculation when virus-induced local lesions were becoming necrotic. RNA synthesis in extracts from leaves infected for 7 days proceeded linearly for about 10 min and required all four ribonucleoside triphosphates and magnesium ions. Enzyme activity was significantly increased by the addition of EGTA to the assay mixture. The polymerase activity was insensitive to actinomycin D, DNase, rifampicin and a-amanitin showing that it was RNA-dependent. The virus-specific RNA-dependent RNA polymerase-template complex in crude extracts from VTMoV-infected leaves and in partially purified preparations was shown to synthesize ds-RNAs of M, about 3.5 X 106 and 0.72 X 106, specific to VTMoV RNAs 1 and 3, respectively. The in vitro synthesis appears to have involved only the elongation of positive strands of RNAs 1 and 3 on their negative strand templates.