Lipid composition of Mycoplasma orale was examined and compared with that of horse serum added to the growth medium. Ratios of cholesterol/cholesterol ester and sphingomyelin/phosphatidylcholine were much higher in M. orale than in the horse serum, indicating the organism incorporates selectively cholesterol and sphingomyelin. A distinct difference between the lipids from the two sources was that in phospholipids of M. orale almost all (greater than 95%) of the fatty acyl residues were saturated whereas nearly half of the residues were unsaturated in horse serum phospholipids. Approximately one third of M. orale phospholipids was phosphatidylglycerol, which was synthesized by the organism as was demonstrated by 32P-labeling experiment. Its acyl residues consisted mainly of C16:0 and were efficiently labeled with 14C-palmitate but not with 14C-acetate. These results clearly indicate the de novo synthesis of phosphatidylglycerol by M. orale is through acylation with exogenous saturated fatty acids. On the other hand, all the phosphatidylcholine and sphingomyelin of M. orale were derived from the medium. The 14C-labeling experiment demonstrates that no fatty acid synthesis takes place nor exogenous fatty acid can be incorporated so efficiently as phosphatidylglycerol, suggesting that extremely high proportion of saturated fatty acyl residues in these phospholipids is the consequence of saturation directed to the acyl chains of the incorporated phospholipids.