As part of an enhanced surveillance programme for pertussis in England and Wales, a real-time PCR service for the detection of Bordetella pertussis was introduced for infants aged ≤6 months admitted to a paediatric intensive care unit or paediatric ward with a respiratory illness compatible with pertussis. Two real-time fluorescent resonance energy transfer hybridization probe LightCycler (Roche Diagnostics) PCR assays were used. One (designed in-house) targeted the pertussis toxin S1 promoter (ptxA-pr), and included an internal process control to test for sample inhibition and reagent performance. The other (already published) targeted the insertion element IS481. The analytical sensitivities of the assays were 100 and 10 fg per reaction for the ptxA-pr and IS481 PCRs, respectively. The ptxA-pr assay was specific for B. pertussis, whilst the IS481 PCR also showed some cross-reactivity with Bordetella holmesii and the type strain of Bordetella parapertussis. From April 2002 to March 2007, 848 samples were received from 774 patients and DNA was extracted. Of 824 samples that were suitable for testing, 183 (22.2 %) had evidence of Bordetella infection (18.9% ptxA-pr and IS481; 3.3% IS481 only), 621 (75.4 %) were negative and 20 (2.4 %) were inhibitory for the PCR. Within the targeted age group of ≤6 months, most patients (130/138) with evidence of Bordetella spp. by PCR were ≤3 months old. The overall percentage increase in laboratory-confirmed cases due to PCR compared with culture for the 5 year period described ranged from 9 to 26% per year (mean 19 %). Real-time PCR is an invaluable tool both for enhanced epidemiological surveillance and for the provision of a rapid diagnosis of pertussis where results can affect patient and contact management.