Analysis of purified Rift Valley fever virus (RVFV) particles demonstrated the presence of three negative-sense RNA genomes, plus three anti-viral-sense RNA segments. The virion-associated anti-viral-sense S segment served as a template for the synthesis of NSs mRNA immediately after infection. NSs protein synthesis also occurred early in infection, suggesting that NSs protein produced early in infection probably has biologically significant roles in virus replication and/or survival in the host. Translation inhibitor treatment of mammalian cells infected with viruses belonging to the Bunyaviridae family generally inhibits viral mRNA synthesis. However, RVFV NSs mRNA synthesis, but not N mRNA synthesis, was resistant to puromycin treatment during primary transcription, pointing to the uniqueness of RVFV NSs mRNA synthesis.