Phagocytic cells exposed to exogenous arachidonic acid (AA) incorporate large quantities of this fatty acid into choline and ethanolamine glycerophospholipids, and into phosphatidylinositol (PtdIns). Utilizing liquid chromatography coupled to MS, we have characterized the incorporation of exogenous deuterated AA ([2H]AA) into specific PtdIns molecular species in human monocyte cells. A PtdIns species containing two exogenous [2H]AA molecules (1‐[2H]AA‐2‐[2H]AA‐glycero‐3‐phosphoinositol) was readily detected when human U937 monocyte‐like cells and peripheral blood monocytes were exposed to [2H]AA concentrations as low as 160 nm to 1 μm. Bromoenol lactone, an inhibitor of Ca2+‐independent phospholipase A2 (iPLA2), diminished lyso‐PtdIns levels, and almost completely inhibited the appearance of 1‐[2H]AA‐2‐[2H]AA‐glycero‐3‐phosphoinositol, suggesting the involvement of deacylation reactions in the synthesis of this phospholipid. De novo synthesis did not appear to be involved, as no other diarachidonoyl phospholipid or neutral lipid was detected under these conditions. Measurement of the metabolic fate of 1‐[2H]AA‐2‐[2H]AA‐glycero‐3‐phosphoinositol after pulse‐labeling of the cells with [2H]AA showed a time‐dependent, exponential decrease in the level of this phospholipid. These results identify 1‐[2H]AA‐2‐[2H]AA‐glycero‐3‐phosphoinositol as a novel, short‐lived species for the initial incorporation of AA into the PtdIns class of cellular phospholipids in human monocytes.