In the presence of an RNA- temperature-sensitive (ts) mutant helper virus, two coronavirus mouse hepatitis virus (MHV) defective interfering (DI) RNAs complemented each other, resulting in the assembly of MHV particles; we used this ability to complement as a means to study coronavirus assembly. One of the two DI RNAs was DIssA, a naturally occurring self-replicating DI RNA encoding N protein and the gene 1 proteins that encode RNA polymerase function; DIssA supports the replication and transcription of other non-self-replicating DI RNAs. The other DI was a genetically engineered DI RNA that encoded sM and M proteins. Coinfection of these two DIs at the nonpermissive temperature for the ts helper virus resulted in replication and transcription of both DI RNAs but not in synthesis of the helper virus RNAs. MHV particles containing DI RNAs, N protein, and M protein, all of which were exclusively derived from the two DI RNAs, were released from the coinfected cells; the amount of sM protein was below the limits of detection. Analyses of DI RNAs with mutations in the two envelope protein genes demonstrated that M and sM proteins appeared to be required for assembly and release of MHV particles that contained DI RNAs and N protein, while S protein was not required for assembly and release of MHV particles.