Comprehensive lipid profiling by mass spectrometry provides comparative data on the relative distribution of individual glycerophospholipids within each of the major classes. Application of this method to the analysis of glycerophospholipid remodeling in murine primary resident peritoneal macrophages (RPMs) during zymosan phagocytosis reveals significant decreases in the levels of every major arachidonic acid (20:4)-containing species of phosphatidylcholine (GPCho) and in selected 20:4-containing phosphatidylinositol (GPIns) and phosphatidylglycerol (GPGro) species. No net changes in 20:4-containing phosphatidylethanolamine (GPEtn) species were detected. Pretreatment of RPMs with LPS resulted in subtle changes in the magnitude and kinetics of the response but had no effect on the overall pattern of zymosan-induced glycerophospholipid remodeling. Inhibition of prostaglandin (PG) synthesis with indomethacin reduced the magnitude of the changes in 20:4-containing diacyl but not alkyl acyl species. Blockade of 20:4 reacylation with thimerosal had no effect on the magnitude of the zymosan-induced changes in GPCho, GPIns, or GPGro species but revealed decreases in the level of alkyl acyl GEtn species. RAW264.7 cells contain much lower levels of phospholipid 20:4 than do RPMs and synthesize PGs poorly in response to zymosan. Pretreatment with granulocyte-macrophage colony stimulating factor, lipopolysaccharide, and interferon-gamma substantially increased the extent of 20:4 mobilization and PG synthesis in these cells. However, under conditions of maximal zymosan-dependent PG synthesis, the only glycerophospholipid that exhibited a significant change was a 20:4-containing plasmenyl GPEtn. These results suggest that GPCho is the major ultimate source of 20:4 that is mobilized in zymosan-stimulated RPMs but that 20:4 mobilization may involve the intermediate turnover of alkyl acyl GPEtn species.