A new phospholipid that can account for up to 40% of the total cellular phospholipid of Rhodopseudomonas sphaeroides has been identified. Purification of the phospholipid was accomplished by column chromatography on silicic acid and diethylaminoethylcellulose followed by preparative thin-layer chromatography. A combination of spectroscopic and chemical techniques were used to identify the unknown phospholipid as an N-acylphosphatidylserine. Infrared spectroscopy revealed the presence of both ester and amide bonds in the phospholipid. Interpretation of the proton nuclear magnetic resonance spectrum of the new phospholipid indicated the presence of three acyl chains per phospholipid and in all other respects was compatible with the proposed structure of the molecule. Chemical studies confirmed the presence of a glycerylphosphorylserine moiety in the molecule and yielded three fatty acyl chains per hydrolyzed phospholipid. The fatty acid composition of the phospholipid was approximately 85% vaccenic acid, 9% stearic acid, 5% palmitic acid, and 1% palmitoleic acid, which is essentially identical with the fatty acid composition of whole cell phospholipid preparations from R. sphaeroides. Chemical synthesis of an N-acylphosphatidylserine from beef brain phosphatidylserine and palmitic anhydride gave a product with characteristics similar to those of the naturally occurring material isolated from R. sphaeroides.