Summary: Some properties of a complement-fixing (CF) antigen for Eaton pleuropneumonia-like organism (PPLO) (Mycoplasma pneumoniae) are described. CF antigen was found to be heat stable and extractable with organic solvents. The properties of the antigen indicated that it either was lipid or was associated with lipid. Raw antigen was extremely anticomplementary (AC). AC activity of whole antigen could be reduced by boiling, while AC activity of solvent-soluble antigens was low. Antigen extracted by either chloroform or ether behaved as a complete antigen by eliciting antibody production in rabbits, while antigen produced by chloroformethanol-aqueous KCl partition (L antigen) was at best poorly immunogenic. For diagnostic purposes L antigen was more specific and demonstrated larger antibody responses than either boiled antigen or ether-extracted antigen. Testing of 40 paired sera from Eaton PPLO isolation-positive patients revealed 36 with 4-fold antibody titer rises, while the 4 others had high antibody in the first sera obtained 7 or more days after onset of illness. Geometric mean titer of first sera was 1:2.3 and of second sera 1:48, indicating an average 20-fold antibody response. Only 12 of 364 (3%) other patients with similar acute respiratory disease who were negative for Eaton PPLO isolation showed 4-fold antibody titer rises with L antigen.