The human promyelocytic leukemia cell line HL60 can be induced to differentiate into mature granulocytes by exposure to Me2SO. [1-14C]Arachidonic acid incubated overnight with these cells was incorporated mainly into membrane phospholipids. Stimulation of these cells with the calcium ionophore, A23187, resulted in a rapid release of esterified arachidonic acid from phosphatidylethanolamine and phosphatidylcholine. The released arachidonic acid was metabolized via both the cyclooxygenase and lipoxygenase pathways into three major hydroxylated products, 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT), 5(S)-hydroxy-6,8,11,14-icosatetraenoic acid (5-HETE), and 5-(S),12(R)-dihydroxy-6,8,10,14-icosatetraenoic acid (leukotriene B). Arachidonic acid was also incorporated into triacylglycerols and phosphatidylinositol. The lipoxygenase product, 5-HETE, was rapidly esterified into cellular lipids. Thirty minutes after ionophore stimulation, 55% of the total 5-HETE synthesized was esterified into phospholipids and 35% incorporated into acylglycerols. In contrast, the other hydroxylated derivatives of arachidonic acid (HHT and leukotriene B) were not incorporated into acylglycerols or phospholipids. Esterification of hydroxylated metabolites of arachidonic acid into membrane phospholipids may serve to regulate a number of granulocyte functions.