We assessed the level and determinants of tetanus-antitoxin (TT)-antibodies in the Dutch population. Additionally, we evaluated the national guidelines for post-exposure prophylaxis. Serum samples and questionnaire data from a cross-sectional, population-based study were obtained from 7903 individuals. Serum antitoxin antibodies were assessed with a multiplex immunoassay. Multivariable linear regression was used to explore factors associated with antibody concentration. The overall seroprevalence was 94% with a geometric mean concentration (GMC) of 0.91 IU/ml. The TT-GMC increased with age in the age-cohorts of 13-23 years, which coincides with the meningococcal C conjugate mass-vaccination in 2002. Lower seroprevalences were found in individuals born before introduction of routine vaccination, first-generation migrants from non-Western countries born before 1984, and conservative Protestants living in the Dutch 'Bible belt'. Only 10% of those eligible for post-exposure prophylaxis were not sufficiently protected against tetanus.