Immunoglobulin M (IgM) and IgG antibodies to Japanese encephalitis virus (JEV) were detected in acute-phase cerebrospinal fluid (CSF) specimens from patients with acute encephalitis by using a solid-phase radioimmunoassay of the antibody capture type. Of 12 patients with JEV infections subsequently proven by hemagglutination inhibition serology, 11 had JEV IgM antibodies, as measured by antibody capture radioimmunoassay, in the first CSF specimen (geometric mean titer, 1:2,500) compared with 0 of 8 patients with acute encephalitis proven not to be due to JEV. Specific IgM anti-JEV activity (units per microgram) was greater in CSF than in parallel serum specimens in all 11 positive cases by more than fourfold on the average (range, 1.4 to 13). Among seven patients with broadly reactive hemagglutination inhibition seroresponses typical of persons previously exposed to other flaviviruses, six had high levels of JEV IgG antibodies (as measured by antibody capture radioimmunoassay) in their acute-phase CSF (geometric mean titer, 1:26,000), whereas in five patients experiencing their first flavivirus infection, JEV IgG antibodies measured by antibody capture radio-immunoassay were either absent (one patient) or weakly reactive (four patients; geometric mean titer, 1:3,200). Specific IgG anti-JEV activity was greater in CSF than in parallel serum specimens in eight of the nine positive cases measured (range, 1.3- to 24-fold). The antibody capture solid-phase immunoassay approach is well suited for detecting specific antibody activity in CSF.