Mouse hepatitis virus (MHV), a member of Coronaviridae, use a unique replication/transcription strategy. The genome is copied into both genome- and subgenome-length negative strands that are used as templates for the synthesis of genomic and subgenomic-length mRNAs. Replicative intermediate/replicative form (RI/RF) containing genome and subgenome-length negative sense RNA accumulate until 5-6 hours postinfection (p.i.), a time when positive viral RNA synthesis reaches a maximum rate. After attaining the maximum rate, positive strands synthesis then declines. We have found that MHV RI/RF were unstable and disappeared after 5-6 hr p.i.. And, most surprisingly of all, putting infected cells at 4°C for as little as 5 minutes caused the RI/RF RNA to disappear. In contrast, RI/RF of Semliki Forest virus (SFV) were stable at 4°C in coinfected cells. If MHV infected cells labeled with 3H-uridine were put at 4°C and then extracted with phenol and chloroform, the counts in RI/RF RNA decreased to less than 10% of that found in cells kept at 37°C. Although not in RI/RF RNA, the negative strands were present in infected cells exposed to 4°C. If viral RNA was allowed to anneal before treatment with RNase, the 3H-uridine in negative sense RNA was recovered as RNase resistant counts. This suggests either that MHV transcription complexes dissociate at 4°C or that exposed to 4°C causes the nascent strands of RNA of transcription complex no longer to be hybridized to their templates. Free negative sense RNA were unable to be fully restored to RI/RF RNA after shift back to 37°C. Viral RNA synthesis resumed, albeit not immediately, when the infected cells are returned to 37°C. But continuous protein synthesis was required. Free negative sense RNA were also detected late in infection or after CHI incubation; but these free negative sense RNA, together RI/RF RNA, eventually declined. These results offered further evidence for unstable character of MHV negative sense templates. Our data also suggests the destabilization of MHV RI/RF RNA is likely to begin with the dissociation of replication complex which lead to the degradation of free negative sense RNA templates later.