The human histiocytic lymphoma U937 cell line contains a rich source of the 85 kDa cytosolic phospholipase A 2 DMSO-di โ erentiated U937 cells were used as a model to investigate the free arachidonic acid release, (cPLA 2 ). the arachidonate distribution and the phospholipid source of arachidonate upon Ca2' ionophore stimulation. A combination of several chromatographic and mass spectrometric techniques was employed in this study. The amount of free arachidonic acid (AA) released upon stimulation, the arachidonate content in total lipids and in each of the phospholipid classes were determined by gas chromatography/mass spectrometry (GC/MS). Glycerophosphoethanolamine (GPE) was found to be the major pool of arachidonate in di โ erentiated human U937 cells (55% ) and glycerophosphocholine (GPC) and glycerophosphoinositol (GPI) contributed 22 and 8% , respectively. Upon Ca2' ionophore stimulation, GPE class lost the largest amount of arachidonate, followed by GPC class. GPI class, however, gained a substantial amount of arachidonate. Most of the arachidonate depleted from GPE and GPC was recovered as free AA, some of which was rapidly esteriรed into GPI species. GC/MS with electron capture negative chemical ionization provided excellent sensitivity for the measurement of arachidonic acid which was derivatized to its pentaรuorobenzyl ester. Intact phospholipid molecular species including the arachidonylcontaining phospholipid species were identiรed using capillary high-performance liquid chromatography/continuous-รow liquid secondary ion mass spectrometry (CF-LSIMS). No speciรcity was found for releasing free AA among the arachidonyl-containing GPE and GPC species upon Ca2' ionophore stimulation. CF-LSIMS provided a sensitive and e โ ective means of detecting intact phospholipid species.