A major neutralizing epitope (here referred to as the T332 epitope) located on the lateral surface of domain III (DIII) of the West Nile virus (WNV) envelope protein has been identified based on the analysis of murine monoclonal antibodies. However, little is known about the humoral immune response against WNV in a natural host or whether DIII in general or the T332 epitope in particular are important targets of neutralizing antibodies in vivo. To characterize the types of antibodies produced during infection with WNV, we studied a group of naturally infected horses. Using immune adsorption assays coupled with the use of virus particles bearing mutations in the T332 epitope, we found that in some animals neutralizing activity against DIII and the T332 epitope was below the limit of detection. In contrast, some animals generated a significant fraction of neutralizing activity to DIII and the T332 epitope. Thus, while antibodies to the T332 epitope did not represent a significant fraction of the total antibody response in the infected animals studied, in some horses, they comprised a significant fraction of neutralizing activity, making this an important but far from dominant neutralizing epitope. Rather, the neutralizing response to WNV generated in infected horses is both variable and polyclonal in nature, with epitopes within and outside of DIII playing important roles.