Docosahexaenoic acid (DHA) circulates in mammals in lipoproteins and bound to serum albumin as a nonesterified fatty acid as well as esterified in lysophosphatidylcholine (lysoPC). 1-Lyso,2-DHA-glycerophosphocholine (GPC) is an unstable isomer because of a primary alcohol at the sn-1 position. To keep DHA at the sn-2 position of lysoPC, its usual position for the corresponding lysoPC to be acylated into PC in tissues, we synthesized 1-acetyl,2-DHA-GPC and confirmed its structure by use of nuclear magnetic resonance (NMR) spectroscopy in comparison with its positional isomer, 1-DHA,2-acetyl-GPC. 1-Lyso,2-DHA-GPC was prepared from 1-stearoyl,2-DHA-GPC by enzymatic hydrolysis and purified by high-performance liquid chromatography. The isomerization of 1-lyso,2-DHA-GPC into 1-DHA,2-lyso-GPC was obtained by keeping the former overnight at room temperature under nitrogen. Both lysoPC isomers were acetylated by acetic anhydride into 1-acetyl,2-DHA-GPC and 1-DHA,2-acetyl-GPC, respectively, and the resulting phospholipids were fully characterized by NMR. In particular, the 1,2 substitution pattern of the acetyl and DHA chains could be easily detected by 2D heteronuclear multibond correlation. We conclude that 1-acetyl,2-DHA-GPC might be considered as a stable form of 1-lyso,2-DHA-GPC for its delivery to tissues, if the latter exhibits acetyl hydrolase activity.