The nucleotide sequences of the envelope protein of the Kamiyama 1 strain of Japanese encephalitis (JE) virus and a passaged mutant (Kamiyama 2 strain) were determined. Two amino acid differences, Ser-Phe at residue 364 and Asn-Ile at residue 367, distinguished Kamiyama 2 from Kamiyama 1. Six neutralization-resistant variants were selected from these two strains using a JE species-specific monoclonal antibody with neutralization and hemagglutination-inhibition reactivities. All variants had a single amino acid substitution at residue 52 and significantly reduced reactivity with other JE species-specific monoclonal antibodies. The variants derived from Kamiyama 2 strain showed reduced virulence in 3-week-old mice after peripheral inoculation but were as virulent as the parent virus when inoculated intracranially. These variants also showed altered early virus-cell interaction but not replication and reproduction in Vero cells. These findings indicate that the mutations at residues 52, 364, and 367 affect early virus-cell interaction in Vero cells and virulence in mice.