Real-time PCR was employed to detect a region of the P1 cytadhesin gene of Mycoplasma pneumoniae in clinical samples. An internal processing control was included that could be co-amplified simultaneously in the same reaction tube. The assay could reproducibly detect 1 × 103 M. pneumoniae organisms ml-1 in clinical samples. There was no amplification of DNA or signal production from 15 other species of human mycoplasmas and 19 other bacterial species. Using a panel of 175 respiratory samples taken from patients with pneumonia of proven aetiology, the sensitivity was found to be 60% and the specificity of the assay 96.7% when compared with serology. This assay is suitable for same-day diagnosis of M. pneumoniae infection and batch processing of respiratory samples for clinical screening.