When HL-60 cells are incubated in media containing 10 microM [3H]arachidonic acid the label is immediately incorporated into both triacylglycerols and phospholipids. About one-half of the cellular tritium was associated with triacylglycerols after 2 h of incubation and this [3H]arachidonate was then transferred to phospholipids as soon as the labeled cells were placed in arachidonate-free media. A technique was devised to analyze the stereospecific distribution of [3H]arachidonate at the three sn-positions of glycerol in order to identify the mechanism(s) responsible for the biosynthesis of the labeled triacylglycerols. [3H]Arachidonate was found to be distributed in nearly equal amounts among all three glycerol positions of the triacylglycerols. In addition, analysis of intact triacylglycerols containing [3H]arachidonate revealed that 24% of the tritium eluted from reverse-phase HPLC with triarachidonoylglycerol. Both of these findings would be expected if a significant portion of the arachidonate-containing triacylglycerols were synthesized de novo. Homogenates prepared from [3H]arachidonate prelabeled HL-60 cells were capable of hydrolyzing the endogenous [3H]arachidonate-containing triacylglycerols to produce mainly free fatty acids and smaller amounts of monoacylglycerols. The relatively small amount of monoacyl- and diacylglycerols produced by the lipolytic activity of the homogenates indicated that [3H]arachidonate was hydrolyzed from all three sn-positions of the [3H]triacylglycerols. This lipase activity had a pH optimum of 4.5 and was associated to a greater extent with the soluble fraction than in the total membrane fraction. Although it is not known whether this lipolytic activity is the same as that expressed in the intact cells, the activity of the cell-free triacylglycerol lipase was of sufficient magnitude to have easily accounted for the decrease in [3H]triacylglycerols that was observed after transfer of the intact HL-60 cells (prelabeled with [3H]arachidonate) to fresh media. The data suggest that transfer of arachidonate from triacylglycerols to phospholipids probably occurs through an acyltransferase utilizing a lysophospholipid and arachidonoyl-CoA.