Two isolates of the murine hepatitis virus (MHV) strain JHM, which differed in their ability to express the nonstructural gene product ns2, were characterized. The MHV Wb3 isolate encodes a 30,000-molecular-weight ns2 protein that can be readily detected in infected cells by using a specific monoclonal antibody, MAb 2A. The MHV Wb1 isolate is a deletion mutant that lacks a functional ns2 gene and the transcriptional signals required for the synthesis of an ns2 mRNA. However, there are no obviously significant differences in the growth of the MHV Wb1 and MHV Wb3 isolates in continuous cell lines or in the synthesis of viral mRNAs or proteins in infected cells. These results demonstrate that the ns2 gene product is not essential for MHV replication in transformed murine cells and suggests that the function of the ns2 gene may only be manifest in vivo.