The use of a 16S rRNA based polymerase chain reaction (PCR) for the detection of Mycoplasma pneumoniae infection was investigated. Sputum samples from 34 patients with respiratory illness and evidence of pneumonia as judged by chest X-ray were analyzed by PCR and microbiological culture. Throat swabs from 14 healthy individuals were used as controls. For serology, an enzyme immunoassay for the detection of immunoglobulin M antibodies and a complement fixation assay were performed. Evidence of Mycoplasma pneumoniae infection was obtained in ten patients (29%), eight of whom were found positive by both PCR and serology. Two of the sputum samples from these eight patients were negative by culture. Of the remaining two patients positive for Mycoplasma pneumoniae, one was positive by PCR and culture but negative by serology, and one was found positive by serology but negative by PCR and culture. Thirteen of the 14 controls were negative by both PCR and serology. One control, however, was negative by serology but positive by PCR, which was probably due to asymptomatic carriage of Mycoplasma pneumoniae. The results of this study indicate the suitability of the PCR for the detection of Mycoplasma pneumoniae in clinical samples as well as its potential value as an additional tool for the diagnosis of infection.