The lack of information on the immunity of adults in Brazil against diphtheria prompted us to analyse sera from 234 blood donors aged 18–61 years (30·3% females and 69·7% males). IgG diphtheria antitoxin levels determined by means of an ELISA, validated by toxin neutralization test in Vero cells, showed that 30·7% (95% CI 25·0–37·1) of the population was fully protected ([ges ]1 IU/ml). The highest percentage of subjects fully protected was in the 31–40 years age group. Most of the subjects with uncertain or no protection (<1 IU/ml) were found in the 18–30 years age group (43·8%, OR 2·18, P=0·01). Antitoxin levels were not influenced by the increase in age. Males were more protected than females (80·5%, OR 0·44, P=0·01). The prevalence of 30% of individuals fully protected against diphtheria in blood donors in Rio de Janeiro supports the fact that immunity to diphtheria among healthy Brazilian adults is inadequate. To avoid diphtheria epidemics in the future the immunity among adults should be raised in the coming years.