The last report on pertussis seroprevalence in Belgium concerned samples collected during 1993–1994. In the context of the Eupert-Labnet WP6 seroprevalence study (comparing sera from 16 European member states), 1500 anonymized leftover diagnostic samples were collected randomly during the second semester of 2012 by the clinical chemistry laboratories of six participating Belgian centres, distributed equally between Flanders, Wallonia and Brussels Capital Region. As suggested by the WP6 organizers, a total of 750 samples (125/centre) were selected from subjects in the 20–29 years age group and 750 samples (125/centre) from subjects in the 30–39 years age group. Anti-PT IgG levels were measured using Virion-Serion ELISA and analysed using predefined cut-off levels. Sixty-one (4%) sera were indicative of an infection in the past 2 years (between 50 and 100 IU/ml) and another 61 (4%) sera had anti-PT IgG antibodies reflecting acute infection (>100 IU/ml). These results highlight the presence of a Bordetella pertussis reservoir in the adult ‘healthy’ Belgian population.