A phosphoglycolipid, presumably identical to glyceryl-phosphoryldiglycosyl diglyceride, is the main component of the membrane glycolipids of Mycoplasma mycoides subsp. capri. It is immunologically active. Anti-phosphoglycolipid antibodies were induced in rabbits by intravenous injection of the flocculated complexes of methylated bovine serum albumin with a mixture of the phosphoglycolipid and the auxiliary lipids, phosphatidyl-choline and cholesterol. The specificities of the antibodies directed against the phosphoglycolipid, are due to both the phosphate and carbohydrate moieties of the lipid molecule. Anti-phosphoglycolipid antibodies were detected in the sera of rabbits intravenously immunized with intact M. mycoides subsp. capri. The intravenous method of immunization was chosen in order to select for a response to surface antigenic determinants. Anti-phosphoglycolipid antibodies specifically reacted with intact organisms and isolated membranes of M. mycoides subsp. capri, as shown by complement fixation and agglutination tests. The antigenic determinants of the phosphoglycolipid are mainly located on the outer membrane surface. It is concluded that the antigenic determinants of the phosphoglycolipid in intact M. mycoides subsp. capri significantly contribute to the surface architecture of mycoplasma membranes.