Incorporation of arachidonic acid into phospholipid molecular species of the human neutrophil was found to be dependent, to a large extent, upon the concentration of arachidonate used during the in vitro incubations. When high concentrations of [3H] arachidonate were employed, only two glycerolipids incorporated label. One glycerolipid was a unique glycerophospholipid characterized by HPLC retention time and fast atom bombardment mass spectrometry as 1,2-diarachidonoyl-sn-glycero-3-phosphocholine. The second and most highly labeled glycerolipid was found to be arachidonoyl triacylglycerol species. Human neutrophils isolated from normal individuals and not previously exposed to arachidonic acid in vitro were found to contain a small but measurable amount of diarachidonoyl-GPC. The dose-dependent increase of diarachidonoyl-GPC and arachidonoyl-labeled triacylglycerol when cells were exposed to increasing concentrations of arachidonic acid implied that these lipid molecular species have the capacity to expand their pools, perhaps in manner regulating levels of endogenous arachidonic acid for further metabolism. These observations point to the importance of the concentration of arachidonic acid employed during in vitro labeling studies.