Envelope glycoprotein (E) prepared from purified Japanese encephalitis (JE) virus was cleaved with cyanogen bromide (CNBr) followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Mice were immunized with 36 kD, 27 kD, and 8 kD bands from CNBr-cleaved and 54 kD band from control specimens. Neutralization test was positive in one out of two anti-54 kD, in none of the anti-36 kD, and all of anti-27 kD and anti-8 kD sera at 1:10 dilution. Geometrical mean ELISA titre was the highest for anti-54 kD followed by anti-36 kD, anti-27 kD, and anti-8 kD sera. Reactivity of these sera to CNBr-cleaved fragments in Western blotting indicated that the 8 kD fragment was a part of the 27 kD but was not included in the 36 kD fragment, while the 27 kD and 36 kD fragments shared an overlapping part. These fragments were located on the E protein by N-terminal amino acid sequencing of each fragment purified by reversed-phase high-performance liquid chromatography and by comparison with the nucleotide sequence of the E protein gene. The 36 kD fragment was located between the third and the ninth methionine and covered most of the N-terminal side of the E protein. In contrast, the 27 kD fragment was located between the fourth and the tenth methionine and included the 8 kD fragment which was situated between the ninth and the tenth methionine near to the C-terminus of the E protein. Denaturation-resistant neutralizing epitope(s) appeared to be present on the 8 kD fragment, but not on the 36 kD fragment.