Abstract Vesicular stomatitis virus synthesizes a leader RNA in addition to the five monocistronic mRNA species in vitro. The leader RNA molecule appears to be an initiated RNA molecule since it is polyphosphorylated at its 5′-terminus. In addition, uv inactivation studies on purified virions suggest that leader RNA is synthesized prior to mRNA synthesis. The leader RNA is coded for at the 3′-end of genome RNA since purified leader RNA will protect the 3′-terminal region of the genome RNA from RNase digestion. Kinetic studies show that leader RNA and mRNA coding for the N protein anneal to 42 S genome RNA at the same rate, indicating that the leader RNA is coded for only once on the 42 S genome RNA. In the absence of UTP, an oligonucleotide is synthesized in vitro which contains the same 5′-terminal sequence as the leader RNA. These results indicate that the synthesis of the leader RNA is the first event during in vitro RNA transcription by vesicular stomatitis virus.