Chlamydia pneumoniae has recently been recognized to cause various human respiratory tract diseases, including pharyngitis, bronchitis and pneumonia. The most common pathogens of respiratory tract infections such as Streptococcus pneumoniae and Haemophilus influenzae are frequently present in middle ear effusions. Therefore, it is possible that C. pneumoniae play a role in otitis media with effusion (OME). Isolation of C. pneumoniae from middle ear aspirates of OME was performed by the culture method using the HeLa 229 or HL cell line. Identification as C. pneumoniae was based on positive staining for inclusions by a species-specific fluorescein-conjugated monoclonal antibody. C. pneumoniae was recovered from 17.8% (13 of 73) of the patients with acute OME, and 7.1% (3 of 42) of those with chronic OME, Moreover, antibodies to C. pneumoniae were measured by the microimmunofluorescence method in 14 patients who yielded the agent, and all of them had detectable antibodies to C. pneumoniae. Evidence of recent or current infection by the organism was found in nine patients on the basis of either on IgM antibody titer of 1:16 or higher, or a four-fold rise in the titer of IgG antibody. The study findings indicate that C. pneumoniae can cause OME and that the organism is a new candidate as an etiological agent of middle ear diseases.