In addition to the spike (S) glycoprotein that binds to carcinoembryonic antigen-related receptors on the host cell membrane, some strains of mouse coronavirus (mouse hepatitis virus [MHV]) express a hemagglutinin esterase (HE) glycoprotein with hemagglutinating and acetylesterase activity. Virions of strains that do not express HE, such as MHV-A59, can infect mouse fibroblasts in vitro, showing that the HE glycoprotein is not required for infection of these cells. The present work was done to study whether interaction of the HE glycoprotein with carbohydrate moieties could lead to virus entry and infection in the absence of interaction of the S glycoprotein with its receptor glycoprotein, MHVR. The DVIM strain of MHV expresses large amounts of HE glycoprotein, as shown by hemadsorption, acetylesterase activity, and immunoreactivity with antibodies directed against the HE glycoprotein of bovine coronavirus. A monoclonal anti-MHVR antibody, MAb-CC1, blocks binding of virus S glycoprotein to MHVR and blocks infection of MHV strains that do not express HE. MAb-CC1 also prevented MHV-DVIM infection of mouse DBT cells and primary mouse glial cell cultures. Although MDCK-I cells express O-acetylated sialic acid residues on their plasma membranes, these canine cells were resistant to infection with MHV-A59 and MHV-DVIM. Transfection of MDCK-I cells with MHVR cDNA made them susceptible to infection with MHV-A59 and MHV-DVIM. Thus, the HE glycoprotein of an MHV strain did not lead to infection of cultured murine neural cells or of nonmurine cells that express the carbohydrate ligand of the HE glycoprotein. Therefore, interaction of the spike glycoprotein of MHV with its carcinoembryonic antigen-related receptor glycoprotein is required for infectivity of MHV strains whether or not they express the HE glycoprotein.