In mouse hepatitis virus (MHV) infected cells, seven species of positive sense single stranded RNA are detected, including genomic-length RNA and six subgenomic-length RNA. These RNAs can serve as mRNAs and form a 3' end coterminal nest set of RNA with same 5' end leader sequence. Corresponding genomic- and subgenomic-length negative sense RNAs are used as templates for mRNA synthesis. Negative sense RNA accumulated rapidly in replicative intermediate/replicative form (RI/RF) RNA in the first 6 hours postinfection (p.i.). After 6 hr p.i., viral RI/RF RNA begun to decline. If 3H-uridine was added at 6 hr p.i., it accumulated in RI/RF RNA and the percentage of labeled negative sense RNA in the RI/RFs steadily increased. Therefore, negative sense RNA in RI/RF RNA made during the first 6 hr were replaced by negative sense RNA made after 6 hr. If true, negative sense RNA in MHV infected cells are unstable and are continuously replaced by newly made ones. When translation was inhibited by cycloheximide (CHI), negative sense RNA synthesis was inhibited abruptly. Positive sense RNA synthesis, on the other hand, continued unabated for 30-60 min before slowly declining. This suggested that loss of positive sense RNA synthesis after CHI might be due, at least in part, to the loss of negative sense RNA templates, in addition to the failure to produce viral transcriptase. After CHI removal, positive sense RNA synthesis returned quickly to normal levels, and there was a burst of incorporation of 3H-uridine into negative sense RNA that lead to about 70% of the total negative sense RNA in RI/RF RNA were newly made. Unlike MHV, alphavirus RI/RF RNA is stable and does not turnover after inhibition of protein synthesis. In cells infected with MHV and superinfected with the alphavirus Semliki Forest virus (SFV), only the MHV RI/RF RNA declined late in infection. Therefore, the loss of RI/RF RNA is specific to MHV. The unstable character we demonstrated here reveals a possible new way for MHV to regulate its replication and transcription.