To determine whether human antibodies specific for WNV recognize the neutralizing epitope on DIII during infection, plasma was obtained from WNV-positive individuals. Samples from convalescent individuals were negative for WNV RNA but positive for neutralizing WNV-specific antibodies. The individuals reported mild systemic illness, though none progressed to severe disease. To determine whether these samples contained antibodies that localized to the neutralizing epitope on DIII, we tested whether E16 Fab or IgG could compete binding to recombinant, wildtype and mutant N394K and K307N forms of DIII ( Supplementary  Fig. 2 online); N394K retains wild-type binding to E16, whereas K307N has markedly reduced binding. E16 equivalently inhibited binding of patient WNV-specific antibodies to wild-type (Fab, 35% ± 8; IgG, 40% ± 12) or N394K DIII (Fab, 32% ± 9; IgG, 34% ± 11), whereas E53 IgG, a WNV-specific monoclonal antibody that recognizes an epitope outside DIII, did not compete binding to wild-type (1% ± 4) or N394K