An increase in pertussis has been observed in several countries over the last decades, especially in adult populations. The seroprevalence of pertussis was determined in a cross-sectional study of the adult population in the Copenhagen area, Denmark, conducted between 2006 and 2008. Specific IgG antibodies against pertussis toxin (PT) were measured in 3440 persons resulting in an age-standardized seroprevalence of 3·0% (95% confidence interval 1·9–4·7) using an IgG anti-PT cut-off of 75 IU/ml. By using antibody decay profiles from longitudinal data the estimated seroincidence was 143/1000 person-years. In contrast, an incidence of 0·03/1000 person-years was estimated from the official data of notified cases during the same period. Of the investigated risk factors, only age and education were significantly associated with pertussis infection. This study indicates that pertussis is highly underestimated in the adult population in Denmark, which has implications for future prevention strategies, including raising the awareness of pertussis.