Highly purified radiolabeled mouse hepatitis virus (MHV) A59 contained a previously overlooked protein which coelectrophoreses with the gene 5b product immunoprecipitated from infected cells. The gene 5b protein is post-translationally acylated. Rabbit antibody raised against a recombinant gene 5b protein expressed in Escherichia coli neutralized viral infectivity in the presence of complement, although not in the absence of complement. Immunofluorescent staining of MHV-infected cells with two anti-peptide antibodies revealed that the gene 5b product is membrane-associated and is transported to the cell surface, findings consistent with the prediction of a membrane-spanning segment in the gene 5b polypeptide. These results suggest strongly that the gene 5b polypeptide represents a new MHV virion envelope protein which is homologous to the TGEV ORF 4 and IBV 3c proteins.