Six recombinant Japanese encephalitis virus (JEV) isolates were recovered from infectious RNAs transcribed by T7 RNA polymerase from molecularly cloned cDNA templates. Three of the recombinant viruses had characteristics similar to the wild-type parent virus, JaOArS982. The other 3 recombinant viruses exhibited an attenuated phenotype in mice. An avirulent recombinant virus, IC47, was characterized and compared with the wild-type parent virus and a virulent recombinant virus, IC37. IC47 produced smaller plaques than parent or IC37 viruses and exhibited no viremia or neuroinvasion in young adult mice inoculated subcutaneously and no mortality when inoculated intracerebrally. IC47 was also immunogenic and protective in the murine model. The probable basis for attenuation, revealed by nucleotide sequence analysis, was a single amino acid substitution at position 138 (Glu to Lys) in the E protein.