Highly pathogenic avian influenza (HPAI) H5N1 virus infections have recently caused unprecedented morbidity and mortality in a wide range of avian species. European Commission directive 2005/744/EC allowed vaccination in zoos under strict conditions, while reducing confinement measures. Vaccination with a commercial H5N2 vaccine with vaccine doses adapted to mean body weight per species was safe, and proved immunogenic throughout the range of species tested, with some variations between and within taxonomic orders. After booster vaccination the overall homologous geometric mean titre (GMT) to the vaccine strain, measured in 334 birds, was 190 (95% CI: 152-236), and 80.5% of vaccinated birds developed a titre of >or=40. Titres to the HPAI H5N1 virus followed a similar trend, but were lower (GMT: 61 (95% CI: 49-76); 61%>or=40). The breadth of the immune response was further demonstrated by measuring antibody titres against prototype strains of four antigenic clades of currently circulating H5N1 viruses. These data indicate that vaccination should be regarded as a beneficial component of the preventive measures (including increased bio-security and monitoring) that can be undertaken in zoos to prevent an outbreak of and decrease environmental contamination by HPAI H5N1 virus, while alleviating confinement measures.