In an attempt to develop a specific serological test for Murray Valley encephalitis (MVE) virus antibodies, a panel of MVE monoclonal antibodies was utilised in defined-epitope blocking ELISA tests. In sera of mice immunised singly and in combinations of MVE, Alfuy (ALF), and Kunjin (KUN) viruses, blocking patterns usually distinguished MVE infections from those of the other flaviviruses. When blocking tests with selected MAbs were applied to 468 flavivirus antibody positive sera collected from human subjects throughout New South Wales, sera with blocking patterns consistent with previous MVE infection were found in 18 subjects. All were long-term residents of areas previously frequented by MVE, and all were of an age to have been exposed to the virus in past epidemics. No such sera were found in subjects living in coastal areas of NSW where MVE has never been reported.