Twenty-one hybridomas producing monoclonal antibodies specific for the E glycoprotein of St. Louis encephalitis (SLE) virus, strain MSI-7, have been isolated. Serologic reactivities were initially determined by cross-reactivity indirect immunofluorescence assays using 22 strains of SLE virus and 8 other related flaviviruses. Four groups demonstrating type-, subcomplex-, supercomplex-, and group-specific reactivity patterns were identified. Analysis of hemagglutination-inhibition (HI) and virus neutralization (N) subdivided the cross-reactivity groups into eight epitopes (E-1a,b,c,d, E-2, E-3, and E-4a,b). The antibodies could detect strain differences between SLE viruses isolated from various geographic areas. Analysis of the spatial arrangements of these epitopes using competitive binding assays with representative antibodies possessing similar binding avidities, indicated that the protein was a continuum of six overlapping domains.