Of 12 neutralizing monoclonal antibodies, 10 localized to the distal lateral surface of DIII, results that are consistent with prior studies that mapped three neutralizing monoclonal antibodies against WNV using in vitro escape variants 15, 30 . Our weakly neutralizing and non-neutralizing monoclonal antibodies did not recognize this epitope but localized to distinct regions. E16 recognized the dominant epitope and neutralized all strains that were tested. Sequence analysis of 124 WNV strains in public databases showed almost complete (98.4-100%) conservation of the contact residues S306, K307, T330 and T332. Only two clinically attenuated lineage II isolates had mutations at these residues. Because of the structural homology among flaviviruses 7, 8, 16 , the analogous amino acids that map to the distal lateral surface of DIII can be readily identified. Although this region is highly variable among flaviviruses, most mutations that abolish binding of virus-specific neutralizing antibodies map here 15, 18, 30, 31 , suggesting the existence of an analogous dominant neutralizing epitope for other flaviviruses. We speculate that successful vaccines against WNV or other flaviviruses should induce potent humoral responses against this neutralizing epitope.