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A membrane fraction was isolated by differential centrifugation from PVXinoculated tobac protoplasts to investigate in vitro viral RNA synthesis. This fraction was capable of catalyzing the in vitro synthesis of a genomic-length (6.4kb) and two subgenomic-length RNAs (2.1 and 0.9kb). The different sensitivities of the RNAs to S1 nuclease at high and low salt concentrations suggested that the synthesized genomic-length RNA was mainly the replicative intermediate RNA (RI) and that the two subgenomiclength RNAs were singlestranded. Most of the synthesized RNAs were shown to be of a positive polarity based on ribonuclease protection assay. Dependent on the exogenous template, the fraction treated with micrococcal nuclease synthesized only the genomiclength RNA but not the subgenomic RNAs. Treating the membrane fraction with protease abolished synthesis of the sub nomic-length RNAs, but not that of genomiclength RNA. The synthesizing activity for the subge length RNAs was released into the soluble fraction after treatment with the detergent Brij 58.Th results suggested that genomic and subgenomic RNAs are replicated at different sites in the memb genomic RNA synthesis occurs inside the membranes, whereas subgenomic RNA synthesis occurs on their surface.(Received February5,1997;AcceptedDecember1