Yellow fever virus (YF) is the prototype member of the Flavivirus genus. Here, we report the successful construction of a full-length infectious cDNA clone of the vaccine strain YF-17D. YF cDNA was cloned into a low-copy-number plasmid backbone and stably maintained in several E. coli strains. Transcribed RNAs had a specific infectivity of 10(5)-10(6) p.f.u. ( micro g RNA)(-1), and the resulting virus exhibited growth kinetics, plaque morphology and proteolytic processing similar to the parental virus in cell culture. This clone was used to analyse the importance of conserved flavivirus RNA sequences and the 3' stem-loop structure in virus replication. The conserved sequences 5'CS and CS1, as well as the 3' stem-loop structure, were found to be essential for virus replication in cell culture, whereas the conserved sequence CS2 and the region containing YF-specific repeated sequences were dispensable. This infectious clone will aid future studies on YF replication and pathogenesis, as well as facilitate the development of YF-17D-based recombinant vaccines.