Chlamydia secrete a genus-specific glycolipid antigen (GLXA) into the supernatant of infected cell cultures. The antigen was detected by utilizing a GLXA-specific monoclonal antibody (89MS30) in a chemiluminometric assay system; some cross-reaction was demonstrated to chlamydia-specific lipopolysaccharide (cLPS). The antigen is released into supernatants of cell cultures 18 h post-infection and increases rapidly through 60 h. Biologically, GLXA is completely nonmitogenic and is negative in the limulus lysate assay. Oxidation analysis demonstrated the epitope was sensitive to periodate oxidation. SDS-PAGE analysis showed marked differences in banding patterns between GLXA and LPS, demonstrating they represent physically very different moieties. Sera from patients with C. pneumoniae react with the antigen. Thus, GLXA may be an important molecule in chlamydial infection and subsequent immune responses.