Laboratory confirmation of pertussis by culture, PCR, or detection of antibody increase in paired sera is hampered by low sensitivity in the later stages of the disease. Therefore, we investigated whether, and at which level, concentrations of immunoglobulin G (IgG) antibodies against pertussis toxin (PT), IgG-PT, in a single serum sample are indicative of active or recent pertussis. IgG-PT, measured by enzyme-linked immunosorbent assay in units per milliliter, was analyzed in 7,756 sera collected in a population-based study in The Netherlands, in the sera of 3,491 patients with at least a fourfold increase of IgG-PT, in paired sera of 89 patients with positive cultures and/or PCR results, and in the sera of 57 patients with clinically documented pertussis with a median follow-up of 1.4 years. We conclude that, independently of age, IgG-PT levels of at least 100 U/ml are diagnostic of recent or active infection with Bordetella pertussis. Such levels are present in less than 1% of the population and are reached in most pertussis patients within 4 weeks after disease onset and persist only temporarily.