The immunoblot patterns of 248 sera, all examined previously by the microimmunofluorescence test (MIF) for species-specific Chlamydia antibodies, were analyzed. Predominant specific antibody activity was directed to the 54 kDa protein of Chlamydia pneumoniae, which was recognized by 93% of sera positive for Chlamydia pneumoniae by MIF but by only 2% of sera positive for Chlamydia trachomatis and negative for Chlamydia pneumoniae and by 3% of sera negative for both Chlamydia pneumoniae and Chlamydia trachomatis. This antigen appears to be specific for Chlamydia pneumoniae. Other Chlamydia pneumoniae-specific protein antigens were recognized far less frequently. Absorption analysis indicated that the 54 kDa protein is located on the surface of the Chlamydia pneumoniae elementary bodies.