A seroepidemiological study was conducted on a representative sample (n = 3944) of the Spanish population to assess the immune status to diphtheria. A total of 1907 men and 2037 women in the 5-59 years age range were stratified by sex and age (5-12, 13-19, 20-29, 30-39, 40-49, 50-59 years). Concentrations of toxin antibodies were measured using a commercial ELISA kit. Samples with titres > or = 0.1 IU/ml were considered to show full protection. Only 26% (95% CI: 25-28%) of the overall population studied was fully protected. The highest number of subjects with uncertain or no protection was found in the 20-39 age group (> 85%). The prevalence of fully protected subjects progressively declined from 51% for the 5-12 year age group, to 14% for the 30-39 year age group, whereas it increased to 20% and 35% for the 40-49 and the 50-59 year age groups, respectively (p < 0.0001). These results stress the need to intensify vaccination programs in adolescents and adults with periodic booster doses of diphtheria toxoid.