In December 2005, the four major Swiss zoos carried out the vaccination of selected zoo birds with the adjuvant inactivated vaccine H5N2 Nobilis influenza. Pre- and post-vaccination antibody titers were determined either by hemagglutination inhibition (HI) test (non-Galliformes) or by enzyme linked immunosorbent assay (ELISA) (Galliformes) at Week 0, 5, 10, and 26 (Day 0-1, 35-36, 70-71, and 182 respectively) to determine the humoral immune response to H5 antigen. After the first vaccination, the overall geometric mean titer of non-Galliformes was 65 (n = 142), which increased to 187 (n = 139) after booster vaccination and dropped to 74 (n = 65) six months after first vaccination. For the Galliformes group, the mean titers were found to be 2.09 at Week 5 (n = 119), 3.24 at Week 10 (n = 113), and 1.20 at Week 26 (n = 39). Within the non-Galliformes, significant differences in geometric mean titers were found among different species representatives. In general, the flamingos (Phoenicopteriformes) showed a strong response to vaccination, reaching a geometric mean titer of 659 at Week 10, while the Sphenisciformes did not show high antibody titers even after booster vaccination, reaching a maximum geometric mean titer of only 65. Based on the antibody titer profiles of all investigated species, we recommend at least annual revaccination for the species that we investigated.