Paired sera, obtained from 88 American and Thai patients hospitalized with presumed Japanese encephalitis virus (JEV), were tested by the standard serologic techniques of hemagglutination inhibition (HI), complement fixation, and plaque reduction neutralization. On the basis of these tests, 35 patients had recent or remote primary JEV infections, while 53 patients had secondary group B arbovirus infections characterized by high cross-reactive antibody titers to JEV and to dengue virus serotypes 1–4. Immunoglobulin M (1gM) HI antibody, isolated from whole serum by sucrose density gradient centrifugation, reacted with JEV but not with dengue viruses in 31 secondary cases, and in 29 primary infection patients. IgM antibody reacted monospecifically with JEV in 60 of 63 patients whose serum contained detectable IgM activity. Moreover, rising JEV IgM antibody titers were found in 6 of 25 patients having fixed or falling whole serum antibody titers. Altogether, 57% of patients were confirmed as having recent JEV infections by rising IgM antibody titers, while only 26% were confirmed by conventional serologic tests. On the basis of these findings more precise serologic criteria have been formulated which utilize results of both whole serum and IgM antibody titrations for the diagnosis of JEV infections.