Diagnosis of Mycoplasma pneumoniae and Chlamydia pneumoniae lower respiratory infections using DNA amplification by polymerase chain reaction (PCR) on throat swab specimens has been reported. In this study we determined the sensitivity of the detection of Legionella pneumophila in simulated throat swab specimens by PCR. Next, we compared the sensitivity and specificity of a single throat swab PCR with the current tests for diagnosis of Legionella spp., M. pneumoniae, and C. pneumoniae in patients with lower respiratory tract infections. Patients' work-up included: (a) throat swab specimen for Legionella spp., M. pneumoniae, and C. pneumoniae PCR; (b) throat swab specimen for C. pneumoniae culture; (c) sputum specimen for L. pneumophila direct fluorescent antibody and culture; (d) urine specimen for L. pneumophila serogroup 1 antigen detection; and (e) serum specimen for L. pneumophila, M. pneumoniae, and C. pneumoniae acute and convalescent antibody titers. A total of 155 patients with lower respiratory infection were enrolled in this prospective study. Throat swab PCR was positive for Legionella spp. in five of the six patients with legionellosis, indicating the presence of this organism in the oropharynx of patients with Legionnaires disease. Mycoplasma pneumoniae PCR was positive in eight of the nine patients with mycoplasma infection. Chlamydia pneumoniae PCR was positive in the two patients with C. pneumoniae infection. None of the other 138 patients with negative PCR had other positive confirmatory tests for respiratory infection by these three organisms (100% specificity). PCR was able to detect 15 of the 17 infected (88.2%). Results of this investigation indicate that PCR on a single throat swab specimen is a rapid, sensitive, and specific test that may greatly simplify the diagnosis of lower respiratory infection caused by Legionella spp., Mycoplasma pneumoniae, or C. pneumoniae.