To obtain insight into mumps immunity 9 years after introduction of vaccination in The Netherlands, antibodies were measured in a national sample (n = 8298) and in clustered religious groups with low vaccine acceptance (n = 254). All sera were tested by indirect ELISA, and agreement with neutralization assay was assessed in a subsample (n = 623). Overall seroprevalence in the adult age groups in the national sample was 96.2% (95% confidence interval 95.4-97.0%). Seroprevalence was somewhat lower in the vaccinated age groups, but still sufficient to maintain herd immunity. After the first dose of vaccine, an increase up to age three years to 93.2% (89.8-96.6%) and a subsequent decline in prevalence to 88.9% (81.7-96.0%) at age 7 years was seen. Seroprevalence in those eligible for two vaccinations was 94.4% (91.3-97.4%). In the religious groups, seroprevalence was generally lower in the age group 1-4 years (30% (18-95%)) than in the national sample, but similar in the older age groups. Seroprevalence as estimated by neutralization assay was only slightly lower for all age groups > or = 1 year. Therefore, the indirect ELISA is a reliable method for measuring mumps virus-specific antibodies in population-based studies. However, to allow for inter-laboratory comparison, international unitage should be developed.