Greater flamingos (Phoenicopterus ruber ruber), an endangered and popular zoo bird species, are susceptible to West Nile virus (WNV) infection, often with a fatal outcome. To determine whether vaccination of greater flamingos produced an immunologic response with measurable antibody titers and to monitor for adverse effects of vaccination on health status, a vaccine trial against WNV infection, using a killed vaccine licensed for use in horses, was performed in 1-month-old flamingo chicks. Fifteen chicks determined to be seronegative for WNV were divided into 2 groups: Group A (n = 8) received 2, 1-ml IM doses of vaccine 3 weeks apart; and Group B (n = 7) received 1, 0.5-ml IM dose, followed by 2, 1-ml IM doses, all given 3 weeks apart. A booster vaccination of 1 ml was administered to all birds 280 days after the initial vaccination series. Antibody titers were measured after the initial immunization and before and 3 weeks after the booster vaccination by plaque-reduction neutralization testing (PRNT). A positive titer at the 90% plaque-reduction cut-off was not detected after initial vaccination or booster vaccination in any of the birds studied, but serum neutralizing activity was detected in 60% of the samples after the booster at 50%–82% plaque reduction at a 1:20 dilution. No adverse effects of vaccination were observed. The lack of a demonstrable antibody response to WNV vaccination in the flamingos may be attributable to the lack of species specificity of the vaccine, poorly developed immune system in 1-month-old flamingo chicks, poor sample handling or storage, or inadequate vaccine dose volume or frequency of administration.