Synthesis and turnover of membrane phospholipids is essential for cell growth and function, and hydrolysis of membrane phospholipid is central to many intracellular signalling mechanisms. Hydrolysis of phosphatidylcholine (PC) is a major signalling mechanism of neutrophils, leukocytes that phagocytose and kill bacteria as part of the innate immune response, generating phosphatidic acid, diacylglycerol and arachidonate-derived lipid second messengers. We describe here the application of tandem MS/MS electrospray ionisation mass spectrometry to the analysis of molecular patterns of PC synthesis by blood neutrophil and lymphocyte cells from healthy volunteers. This technique combined incorporation of the headgroup choline, methyl-labelled with deuterium (methyl-d₉-choline), with precursor scans of diagnostic labelled and native fragment ions. The technique was very sensitive, permitting detection of d₉ enrichment <0.01%. Results showed that the two different cell types maintained distinct molecular species compositions of PC, even though they were exposed to the same nutrient supply in blood. Moreover, while the pattern of lymphocyte PC synthesis directly mirrored composition, the fractional synthesis of arachidonoyl (C20∶4,n-6)-containing PC species in neutrophils was greatly enhanced compared with composition. This increased turnover of arachidonoyl species in neutrophils may be related to the active synthesis of eicosanoids and other arachidonoyl-derived mediators in this cell type.