Four new temperature-sensitive (ts) mutants of Japanese encephalitis virus (JEV) isolated from porcine kidney cells persistently infected with JEV and seven previously isolated ts mutants were studied. Of the eight mutants tested, five mutants ts1, ts14, ts36, ts48 and ts71, were thermolabile. Analyses of virus induced intracellular polypeptides revealed that with majority of the ts mutants, when grown at restrictive temperature, the viral proteins were quantitatively affected. All the five ts mutants tested for mouse virulence showed attenuation in infant mice by the intracerebral route. Two ts mutants, ts36 and ts48, escaped neutralization with two anti-JEV envelope protein specific monoclonal antibodies, however, these escape mutants reacted efficiently with the same monoclonal antibodies in antigen capture ELISA.