Hybridoma cell lines producing monoclonal antibodies to the JHM strain of mouse hepatitis virus-4 (MHV-4) were established. By indirect immunofluorescence and immune precipitation, monoclonal antibodies of three viral polypeptide specificities were characterized. Monoclonal antibodies to nucleocapsid reacted in the cytoplasm of infected cells and precipitated the 60,000d nucleocapsid polypeptide (VP-4) of MHV-4. Other monoclonal antibodies reacted both in the cytoplasm and on the surface of infected cells and were found to precipitate the 170,000d viral glycoprotein (GP-1). A third set of monoclonal antibodies reacted both in the cytoplasm and on the surface of infected cells and precipitated the 25,000d viral glycoprotein (GP-5) and its precursor VP-6 (23,000d). AntiGP-1 alone had direct neutralizing activity for MHV-4 virus, while in the presence of complement both anti-GP-1 and anti-GP-5 neutralized virus. Only anti-GP-1 had the ability to inhibit the spread of infection due to fusion in L241 cells. Thus, the viral glycoprotein GP-1 likely contains both the attachment and fusion activities of MHV-4.