Objectives: To develop and evaluate a novel method for simultaneous identification of 12 potential bacterial pathogens in children with community-acquired pneumonia. Methods: A multiplex PCR-based reverse line blot (mPCR/RLB) assay was developed, to identify 12 respiratory bacterial pathogens, namely Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Moraxella catarrhalis, Haemophilus influenzae, Haemcphilus influenzae type b, Bordetella pertussis, Klebsiella pneumoniae, Legionella pneumophila, Mycobacterium tuberculosis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and single (uniplex) PCRs were used for S. pneumoniae and H. influenzae only In a preliminary evaluation, we compared the results of mPCR/RLB with those of single (uniplex) PCRs and culture of nasopharyngeal aspirates (NPAs) from 100 children under 5 years, admitted to Beijing Children's Hospital between October 2004 and May 2005, with pneumonia. Results: Reference strains and clinical isolates of all 12 target species were correctly identified by mPCR/RLB. Potential pathogens were isolated from one blood culture and 26% of respiratory cultures. One or more pathogens were identified in 70% of respiratory specimens-by mPCR/RLB in 63%, uPCR only in another 3%, culture only in 2%, and culture plus uPCR in 2%. The species most commonly identified were S. pneumoniae (54%) and H. influenzae (38%, including type b, 4%). Cultures were not performed for B. pertussis, M. tuberculosis, C. pneumoniae or M pneumoniae but each was identified by mPCR/RLB in between one and four specimens. Two or more potential pathogens were identified in 35% of specimens. Ten of 14 S. pneumoniae isolates belonged to serotypes represented in the 11-valent pneumococcal conjugate vaccine. Conclusions: The mPCR/RLB assay is a sensitive tool for identification of respiratory pathogens, including mixed infections and bacteria requiring special culture methods.