Real-time PCR assays based on the LightCycler technology were developed for individual (simplex PCR) and simultaneous (duplex PCR) detection and discrimination of Bordetella pertussis and Bordetella parapertussis in clinical samples. The assays were evaluated with 113 specimens from patients with and without symptoms of pertussis. Results were compared to those from conventional culture and TaqMan real-time PCR. The analytical sensitivity ranged from 0.1 to 10 CFU for B. pertussis and B. parapertussis, and intra- and interassay variations were less than 7%. Results were available within 2 h. With the simplex format, 21 of 100 samples from patients with clinical symptoms of pertussis were positive for B. pertussis and/or B. parapertussis. With the duplex format, 18 of 100 samples were positive. LightCycler PCR increased the diagnostic sensitivity over that of culture by 2.0-fold (duplex PCR) (P = 0.08) to 2.3-fold (simplex PCR) (P = 0.02). Our data suggest that duplex PCR in this format showed good analytical sensitivity but lost some sensitivity on clinical samples compared with the simplex format.