Japanese encephalitis still occurs in endemic and epidemic forms over a wide area of Asia. Although the vaccine against Japanese encephalitis virus (JEV) is widely used, no antiviral drug has been reported. We used several different kinds of furanonaphthoquinone derivatives and found antiviral activity against JEV. Especially, 2-methylnaphtho[2,3-b]furan-4,9-dione (FNQ3) indicated the highest antiviral activity, followed by 2-(1-hydroxyethyl)-, 5(or 8)-hydroxy-, and 2-methyl-5(or 8)-hydroxy-analogs of naphtho[2,3-b]furan-4,9-dione. In the presence of 3 microg/ml FNQ3, the virus yields in Vero cells were 2 x 10(5) PFU/ml at 24 h after infecting with the virus and 10% of the control level. Western blot analysis using anti-E rabbit sera or anti-NS3 showed that the expression of viral proteins was inhibited by treatment with FNQ3. In addition, Northern blot analysis indicated that the appearance of JEV-RNA was also inhibited by FNQ3. These results suggest that FNQ3 inhibits JEV replication through viral RNA and protein synthesis.