Truncated dengue-4 E protein was produced as a fusion protein in insect cells using a baculovirus expression vector to examine its usefulness as a diagnostic antigen. A peroxidase-anti-peroxidase (PAP) staining method was used to examine the immunoreactivity of the antigenic determinants in recombinant virus-infected Sf-9 cells with human sera obtained from dengue (DEN) and Japanese encephalitis (JE) endemic areas (41 sera from DEN patients and 39 sera from JE patients or individuals with high JE-antibody titers). The expressed E protein, in which one-third of the carboxy-terminal end was deleted, reacted with sera from DEN patients, but it failed to react or responded only faintly with sera from JE patients. The antibody titers obtained by the staining method correlated with those obtained by enzyme-linked immunosorbent assay (ELISA) (r = 0.64, P < 0.01). Calculation of the ratio (R) of the titer obtained by the PAP staining method to the ELISA titer can clearly differentiate DEN antibody from JE antibody (high R values in DEN sera and low R values in JE sera). The recombinant protein would be especially useful for diagnostic purposes in regions where DEN and JE viruses co-circulate.