Respiratory disease caused by atypical bacteria remains an important cause of morbidity and mortality for adults and children, despite the widespread use of effective antimicrobials agents. Culture remains the "gold standard" for the detection of these agents. However, culture is labor-intensive, takes several days to weeks for growth, and can be very insensitive for the detection of some of these organisms. Newer singleplex PCR diagnostic tests are sensitive and specific, but multiple assays would be needed to detect all of the common pathogens. Therefore, we developed the Pneumoplex assays, a multiplex PCR-enzyme hybridization assay (the standard assay) and a multiplex real-time assay to detect the most common atypical pathogens in a single test. Primer and probe sequences were designed from conserved regions of specific genes for each of these organisms. The limits of detection were as follows: for Bordetella pertussis, 2 CFU/ml; for Legionella pneumophila (serotypes 1 to 15) and Legionella micdadei, 9 and 80 CFU/ml, respectively; for Mycoplasma pneumoniae, 5 CFU/ml; and for Chlamydia (Chlamydophila) pneumoniae, 0.01 50% tissue culture infective doses. Recombinant DNA controls for each of these organisms were constructed, and the number of copies for each DNA control was calculated. The Pneumoplex could detect each DNA control down to 10 copies/ml. The analytical specificity demonstrated no cross-reactivity between 23 common respiratory pathogens. One hundred twenty-five clinical bronchoalveolar lavage fluid samples tested by the standard assay demonstrated that the Pneumoplex yielded a sensitivity and a specificity of 100 and 98.5%, respectively. This test has the potential to assist clinicians in establishing a specific etiologic diagnosis before initiating therapy, to decrease hospital costs, and to prevent inappropriate antimicrobial therapy.