Murine hepatitis viruses provide excellent animal models for the study of virus-induced diseases of the central nervous system and gastrointestinal tract. Several studies have indirectly provided evidence that the spike glycoprotein (S) of these coronaviruses bears determinants for pathogenesis and the induction of protective immunity. In order to directly evaluate the immunogenicity of this protein, it was purified by affinity chromatography with an in vitro neutralizing and in vivo protective monoclonal antibody which immunoprecipitated the 180-kDa spike glycoprotein of the neurotropic A59 strain of murine hepatitis virus (MHV-A59). Mice immunized twice with approximately 1 micrograms of purified S in Freund's adjuvant developed high titers of neutralizing and fusion inhibiting antibodies, even though the protein was at least partially denaturated after elution from the affinity column. Moreover, these mice were protected from lethal encephalitis when challenged intracerebrally with 10 LD50 of MHV-A59. This study provides a direct demonstration of the importance of the coronavirus spike glycoprotein in the induction of a protective immune response.