To identify the molecular determinants for attenuation of wild-type Japanese encephalitis (JE) virus strain SA14, the RNA genome of wild-type strain SA14 and its attenuated vaccine virus SA14-2-8 were reverse transcribed, amplified by PCR and sequenced. Comparison of the nucleotide sequence of SA14-2-8 vaccine virus with virulent parent SA14 virus and with two other attenuated vaccine viruses derived from SA14 virus (SA14-14-2/PHK and SA14-14-2/PDK) revealed only seven amino acids in the virulent parent SA14 had been substituted in all three attenuated vaccines. Four were in the envelope (E) protein (E-138, E-176, E-315 and E-439), one in non-structural protein 2B (NS2B-63), one in NS3 (NS3-105), and one in NS4B (NS4B-106). The substitutions at E-315 and E-439 arose due to correction of the SA14/CDC sequence published previously by Nitayaphan et al. (Virology 177, 541-552, 1990). The mutations in NS2B and NS3 are in functional domains of the trypsin-like serine protease. Attenuation of SA14 virus may therefore, in part, be due to alterations in viral protease activity, which could affect replication of the virus.