A full-length cDNA clone of the potato virus Y (PVY) genome was obtained after cloning difficulties in Escherichia coli were overcome. These difficulties were mainly due to the expression of the CI gene from upstream prokaryotic promoter-like elements within the PVY genome. To overcome this problem, PVY cDNA was maintained in two subclones which were ligated before infection. A plasmid in which these two fragments were contained could be propagated in some E. coli strains but was unstable and yielded only low levels of plasmid DNA. Replacement of the 35S promoter by the SP6 promoter slightly increased the stability of the plasmid and its RNA transcripts were infectious when capped with m7G(5')ppp(5')G. Using two inoculation methods (mechanical or biolistic) the cDNA and its RNA transcript proved infectious on three Nicotiana species and on Solanum tuberosum.