An in vitro system is described which supports the synthesis of vesicular stomatitis virus (VSV) negative-strand RNA. The major components of this system are (i) an mRNA-dependent rabbit reticulocyte lysate to carry out cell-free protein synthesis, (ii) the five VSV mRNAs to program VSV-specific protein synthesis, and (iii) nucleocapsids containing positive- and negative-strand genome-length RNA. The protein products synthesized in the system in response to addition of saturating amounts of the five VSV mRNA's included polypeptides which comigrated in acrylamide gels with the five VSV proteins. Approximately 200 pmol of protein per ml was synthesized during a 90-min reaction. The RNA products synthesized in the system included all five of the VSV mRNA's and, in addition, negative-strand, genome-sense RNA. All of the negative-strand RNA, which represented 2 to 5% of the total RNA product synthesized in vitro, banded in CsCl at the position of nucleocapsids. All of the mature mRNA's made in the system pelleted in CsCl. This technique allowed a clear separation of negative-strand product from the mRNA products and facilitated further analysis of the negative-strand product. The amount of negative-strand product produced in the system was shown to be a function of the amount of concurrent protein synthesis in the system. An increase in the level of protein synthesis led to an increase in the amount of negative-strand RNA synthesized, whereas inhibition of protein synthesis by cycloheximide resulted in a 70% inhibition of negative-strand synthesis. In contrast to the negative-strand RNA product, the amount of transcriptive product was decreased by 50% in the presence of maximum levels of viral protein synthesis. This inhibition was reversed by adding cycloheximide. Characterization of the negative-strand product by Northern blot analysis demonstrated that negative-strand product was being synthesized which hybridized to all five of the VSV mRNA's and, hence, that product representing all of the VSV cistrons was being made. This in vitro system offers an opportunity to study factors involved in the promotion of VSV genome replication as well as those responsible for the regulation of transcription.