Test vaccines comprised of inactivated water-in-oil emulsions containing various antigen levels were prepared using a non-pathogenic H5N1 avian influenza (AI) virus, Alduck/Hokkaidol Vac-1/04 (H5N1). The potencies of these test vaccines were evaluated by two experiments. In the first experiment, the triangular relationship among the antigen levels of test vaccines, the hemagglutination inhibition (HI) antibody response, and the protective effect against challenge with a highly pathogenic avian influenza (HPAI) virus, A/chicken/Yamaguchi/7/04 (H5N1), was confirmed. Then lasting immunity of chickens after a single-shot vaccination was confirmed in the second experiment. As a result, complete protection after the challenge was observed in chickens immunized by test vaccines with an antigen level of 160 HA units/dose or higher. Thus, it was ascertained that the minimum antigen level in the AI vaccine was 160 HA units/dose, and the minimum HI antibody titer that could protect chickens from HPAI virus infection-related death was considered to be 1:16. Dose-dependent HI antibody responses were observed in chickens after the vaccination. Thus, 640 HA units/dose was thought to be similar to the optimal antigen level. Alternatively, the HI antibody titers of chickens, injected with the vaccine containing 640 HA units/dose, were maintained at 1:181 or higher for 100 weeks after the single-shot vaccination.