Atypical pathogens such as Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae are an important cause of community-acquired pneumonia. The available detection methods (culture and serology) either lack sensitivity or give only a retrospective diagnosis. In order to improve their detection and quantification in respiratory samples, a real-time multiplex PCR, performed in two separate reactions, was developed for these three pathogens. The comparison of multiplex real-time and conventional PCR assay on 73 respiratory specimens showed an overall agreement of 98.3%, corresponding to 95.8%, 100% and 100% agreement for C. pneumoniae, L. pneumophila and M. pneumoniae, respectively. Clinical application of this multiplex real-time PCR was done on 40 respiratory samples from 38 patients with respiratory tract infections. Of 19 serology-positive patients, 14 were confirmed by the multiplex real-time PCR to be infected by either one of the three pathogens. All samples from serology-negative patients were negative with the multiplex real-time PCR.