A mass rabies vaccination of dogs has been conducted annually in Japan over the last 60 years. To assess both current levels of rabies virus-neutralizing antibody (VNA) in dogs and the rationale for current vaccination procedures, we used a rapid fluorescent focus inhibition test to determine VNA levels in 756 dogs that had visited animal hospitals in Japan. We found that 51.1% of the dogs that had received 1 rabies vaccination had protective VNA levels (≥0.5 IU/ml) with a geometric mean of 0.61 IU/ml. In contrast, 97.8% of the dogs that had been vaccinated at least twice had protective VNA levels with a geometric mean of 7.86 IU/ml. Furthermore, 97.9-100% of the dogs vaccinated at least twice retained protective VNA levels into the second year after the last vaccination. Although VNA levels in the dogs vaccinated at least twice tended to decline 2 years after the last vaccination, 78.9% retained protective VNA levels. Thus, the current rabies vaccination schedule provides adequate protection, but the registration system and vaccination schedule needs to be improved to ensure that increased numbers of dogs are vaccinated against rabies.