A real-time PCR assay based on the TaqMan technology was developed for the detection of Bordetella pertussis and B. parapertussis in clinical samples. The assay was evaluated with 182 specimens from 153 patients with and without symptoms of pertussis. The analytical sensitivity ranged from 0.1 to 10 cfu for B. pertussis and B. parapertussis, respectively, and diagnostic sensitivity was 94.1% when culture was used as a reference. No sample from a patient without symptoms of pertussis was positive in PCR. Twenty-four of 28 patients who were negative by culture and positive by PCR assay met the CDC clinical case definition for pertussis; the remaining four patients had paroxysms of shorter duration. Intra- and inter-assay variation were <5% and results were available within 4 h.