West Nile virus (WNV) causes morbidity and mortality in humans, horses, and in more than 315 bird species in North America. Currently approved WNV vaccines are designed for parenteral administration and, as yet, no effectiveoral WNV vaccines have been developed. WNV envelope (E) protein is a highly antigenic protein that elicits the majority ofvirus-neutralizing antibodies during a WNV immune response. Leghorn chickens were given three vaccinations (each 2 wk apart) of E proteinorally (20 microg or 100 microg/dose), of E protein intramuscularly (IM, 20 microg/dose), or of adjuvant only (control group) followed by a WNV challenge. Viremias were measured post-WNV infection, and three new enzyme-linked immunosorbent assays were developed for quantifying IgM, IgY, and IgA-mediated immune response of birds following WNV infection. WNV viremia levelswere significantly lower in the IM group than in both oral groups and the control group. Total WNV E protein-specific IgY production w assignificantly greater, and WNV nonstructural 1-specific IgY w as significantly less, in the IM group compared to all other treatment groups.The results of this study indicate that IM vaccination of chickens with E protein is protective against WNV infection and results in a significantly different antibody production profile as compared to both orally vaccinated and nonvaccinated birds.