Arachidonic acid (AA) incorporation into and release from cellular glycerolipids are thought to be crucial events for the regulation of eicosanoid biosynthesis in inflammatory cells. The goal of our study was to determine the distribution of AA in the lipid pools of human lung macrophages (HLM) isolated from human lung parenchyma and to define the changes in AA pools occurring during cell activation. Mass spectrometry analysis indicated that the major pools of AA in HLM were located in phosphatidylethanolamine (PE), phosphatidylcholine (PC), and triglycerides (TG), and, to a lesser extent, in phosphatidylinositol/phosphatidylserine (PI/PS) and in a phospholipid similar to bis-(monoacylglyceryl)-phosphate (BMP). Exogenous AA was initially incorporated into TG and subsequently distributed within phospholipids. After 24 h of labeling, the distribution of exogenous AA in glycerolipid classes closely mimicked that of endogenous AA. Under these equilibrium conditions, HLM released 18.7 and 20.2% of cellular AA when stimulated with TPA or A23187, respectively. Free AA was the major product released by TPA- or A23187-stimulated HLM. However, A23187 but not TPA also induced the formation of leukotriene B4 and 5-HETE. AA was released from PC > PI/PS > or = PE > BMP. In contrast to phospholipids, the amount of AA in TG increased 15 to 90 min after cell activation and returned to prestimulation levels after 180 min. These data indicate that AA is stored in several glycerolipid pools of HLM. Among these pools, BMP is unique of macrophages and TG are particularly enriched in AA in HLM. During the early stage of cell activation, phospholipids act as a source of AA, whereas TG function as a reacylation pool for AA released from phospholipids. These data indicate that TG and phospholipid pools have a differential role in the control of free AA levels generated during HLM activation.