AbstractSince the carbonyl stretching modes of membrane phospholipids reflect molecular perturbations and reorganizations at the bilayer interface region, a definitive assignment is desired of the vibrations associated with the carbonyl transitions at either the sn‐1 or sn‐2 lipid chain position. Ether glycerol phospholipids containing one alkyl ether chain and one acyl chain, with the carbonyl group in either the sn‐1 or sn‐2 chain, provide lipid systems for establishing these assignments. rac‐1‐0‐Hexadecyl‐2‐palmitoyl‐sn ‐glycero‐3‐phosphorylcholine yields a carbonyl stretching mode at 1716 cm−1, while the carbonyl mode for 1‐palmitoyl‐2‐0‐hexadecyl‐sn ‐glycero‐3‐phosphorylcholine shifts to 1737 cm−1, By eliminating the gauche bond at the α‐carbon position of the sn‐2 acyl chain in 1‐0‐hexadecyl‐2‐acetoyl‐sn‐glycero‐3‐phosphorylcholine, the lower frequency carbonyl fundamental reverts to a higher frequency (1739 cm−1) characteristic of a nearly trans conformation about the C2C1 bond in the ester linkage.