Millimolar concentrations of guanidine hydrochloride (GuHCl) are known to inhibit the replication of many plant and animal viruses having positive-sense RNA genomes. For example, GuHCl reversibly interacts with the nucleotide-binding region of poliovirus protein 2C(ATPase), resulting in a specific inhibition of viral negative-sense RNA synthesis. The use of GuHCl thereby allows for the spatiotemporal separation of poliovirus gene expression and RNA replication and provides a powerful tool to synchronize the initiation of negative-sense RNA synthesis during in vitro replication reactions. In the present study, we examined the effect of GuHCl on mammalian orthoreovirus (MRV), a double-stranded RNA (dsRNA) virus from the family Reoviridae. MRV growth in murine L929 cells was reversibly inhibited by 15 mM GuHCl. Furthermore, 15 mM GuHCl provided specific inhibition of viral dsRNA synthesis while sparing both positive-sense RNA synthesis and viral mRNA translation. By using GuHCl to provide temporal separation of MRV gene expression and genome replication, we obtained evidence that MRV primary transcripts support sufficient protein synthesis to assemble morphologically normal viral factories containing functional replicase complexes. In addition, the coordinated use of GuHCl and cycloheximide allowed us to demonstrate that MRV dsRNA synthesis can occur in the absence of ongoing protein synthesis, although to only a limited extent. Future studies utilizing the reversible inhibition of MRV dsRNA synthesis will focus on elucidating the target of GuHCl, as well as the components of the MRV replicase complexes.