The attenuated SA-14-14-2 strain of Japanese encephalitis (JE) virus has been used to immunize people in the People's Republic of China. Oligonucleotide fingerprints of the parent SA-14 and vaccine strain indicate that multiple genetic changes occurred during attenuation of the virus. We have cloned and sequenced the genomes of both the virulent SA-14 and attenuated SA-14-14-2 viruses to define molecular differences in the genomes. Forty-five nucleotide differences, resulting in 15 amino acid substitutions, were found by comparing sequences of the SA-14 and SA-14-14-2 genomes. Transversion of U to A occurred at position 39 in the 5'-noncoding region of SA-14-14-2 and another SA-14 vaccine derivative SA-14-5-3. A single nucleotide change in the capsid gene of SA-14-14-2 altered a single amino acid which changed its predicted secondary structure. A silent nucleotide change was found in the prM gene sequence and the M-protein was unchanged. There are seven nucleotide differences, resulting in five amino acid changes, in the E glycoprotein sequence of the two viruses. Nine amino acid differences were found in the nonstructural proteins of SA-14 and SA-14-14-2: one in NS2A, two in NS2B, three in NS3, one in ns4a, and two in NS5. A single nucleotide change at position 10,428 in the 3'-noncoding region is vaccine virus-specific. The nucleotide and deduced amino acid sequences of the vaccine strain SA-14-14-2, the parent virus SA-14, and virulent strains JaOArS982 and Beijing-1 have been compared and are highly conserved.