Yellow fever (YF) virus is the prototype member of the flavivirus family, a diverse group of human and animal pathogens. A live-attenuated strain of YF virus, called 17D, has been used successfully for human vaccination for more than 50 years. In this report we describe the construction of full-length YF 17D cDNA templates that can be transcribed in vitro to yield infectious YF virus RNA. Because of the instability of full-length YF cDNA clones and their toxic effects on Escherichia coli, we developed a strategy in which full-length templates for transcription were constructed by in vitro ligation of appropriate restriction fragments. The YF virus recovered from cDNA was indistinguishable from the parental virus by several criteria. This system should facilitate the molecular genetic analysis of flavivirus replication and attenuation and may allow YF 17D to be used as a carrier for immunologically important epitopes from other disease agents.