This study describes a real-time PCR assay for the detection and identification of Bordetella pertussis and Bordetella parapertussis. The assay is based on amplification of a fragment from the repeat sequence regions IS481 and IS1001 found in B. pertussis and B. parapertussis, respectively, with subsequent species identification by melting curve analysis using SYBR Green chemistry. Discrimination between the two species was straightforward, as the corresponding melting points showed a significant difference of 7 degrees C. The assay was evaluated first with reference strains and retrospective human clinical samples, and then prospectively with 132 human clinical specimens received between March 2003 and December 2005. The assay allowed the rapid detection of 22 positive clinical samples, of which 15, including one fatal case, were not identified by standard culture techniques. The new assay was sensitive and specific, and can be implemented easily using any real-time PCR apparatus.