The antigenic properties of nine wild-type Japanese encephalitis viruses isolated in Taiwan during 1990–1994 were investigated by comparison with two inactivated vaccine strains (Beijing-1, Nakayama-NIH). All of the nine Taiwanese isolates were found to induce higher cytopathology in Vero cells but showed similar mouse virulence as the two vaccine strains. Antigenic characterization using six E protein-specific monoclonal antibodies shows two of the nine wild-type isolates (i.e. CH1949 and CH2195) presented different antigenic properties of hemagglutination inhibition and plaque reduction neutralization. The E-protein gene nucleotide sequences of CH1949 and CH2195 were determined and compared with other published sequences of the two vaccine strains and other 19 Asian/Taiwanese isolates. Phylogenetic tree analysis indicates these two wild-type Taiwanese isolates are more distant from the two vaccine strains.