Cells of the human promyelocytic leukemia cell line HL-60 as well as HL-60 granulocytes induced in vitro by retinoic acid were examined for lipid composition. One of our original aims was to clarify how human granulocyte (differentiated HL-60 cells) synthesized enough precursors of lipid mediators, such as prostaglandins and/or platelet activating factor. Comparison studies yielded the following results. 1) After granulocyte differentiation, total phospholipid of HL-60 cells decreased to about 70% of that of untreated cells, while the content of triglyceride increased to about 200% of the original level. 2) The subclass composition of ethanolamine-containing glycerophospholipid was greatly altered during differentiation; 1-alkenyl-2-acyl glycerophosphoethanolamine (GPE) increased to 166% of that in the untreated cells, while 1,2-diacyl GPE decreased to 46% of the original value. The resultant profile became very similar to that of human peripheral polymorphonuclear leukocytes. 3) During differentiation, the amount of arachidonic acid stored in both phospholipid and triglyceride of retinoic acid-treated HL-60 cells significantly increased. Its distribution was also modified; arachidonic acid in 1,2-diacyl GPE decreased to 63%, while those of 1-alkenyl-2-acyl GPE, choline-containing glycerophospholipids, and phosphatidylinositol increased to 169, 154, and 153%, respectively. These results suggested that the regulatory mechanism of lipid turnover in HL-60 cells was modified during retinoic acid-induced granulocyte differentiation. The alterations were not enough to explain fully the capability of differentiated HL-60 cells to produce lipid mediators upon stimulation, but they were probably one of the factors that regulate these reactions.