Pepino mosaic virus (PepMV)-infected tomato plants were used to develop an in vitro template-dependent system for the study of viral RNA synthesis. Differential sedimentation and sucrose-gradient purification of PepMV-infected tomato extracts resulted in fractions containing a transcriptionally active membrane-bound RNA-dependent RNA polymerase (RdRp). In the presence of Mg(2+) ions, (32)P-labelled UTP and unlabelled ATP, CTP, GTP, the PepMV RdRp catalysed the conversion of endogenous RNA templates into single- and double-stranded (ds) genomic RNAs and three 3'-co-terminal subgenomic dsRNAs. Hybridisation experiments showed that the genomic ssRNA was labelled only in the plus strand, the genomic dsRNA mainly in the plus strand and the three subgenomic dsRNAs equally in both strands. Following removal of the endogenous templates from the membrane-bound complex, the purified template-dependent RdRp could specifically catalyse transcription of PepMV virion RNA, in vitro-synthesized full-length plus-strand RNA and the 3'-termini of both the plus- and minus-strand RNAs. Rabbit polyclonal antibodies against an immunogenic epitope of the PepMV RdRp (anti-RdRp) detected a protein of approximately 164kDa in the membrane-bound and template-dependent RdRp preparations and exclusively inhibited PepMV RNA synthesis when added to the template-dependent in vitro transcription system. The 300 nucleotides long 3'-terminal region of the PepMV genome, containing a stretch of at least 20 adenosine (A) residues, was an adequate exogenous RNA template for RdRp initiation of the minus-strand synthesis but higher transcription efficiency was observed as the number of A residues increased. This observation might indicate a role for the poly(A)-tail in the formation and stabilisation of secondary structure(s) essential for initiation of transcription. The template-dependent specific RdRp system described in this article will facilitate identification of RNA elements and host components required for PepMV RNA synthesis.