Rat peritoneal macrophages activated by ionophore A 23187, were labelled after introduction in the culture medium of 1-0-stearoyl 2-0-[3H] arachidonylglycero-3-phosphocholine (as unique source of tritiated arachidonic acid), or [3H] arachidonic acid which was esterified by cells in phospholipids and triglycerides or remained non esterified. With either cell-labelling method, stimulated macrophages produced tritiated nonesterified fatty acids and eicosanoids which were isolated from cell and medium lipids. When introduced into the culture medium at 1, 5 or 10 microM, the membrane phospholipid analogue 1,2 di-O-hexadecylglycerophosphocholine (dihexadecyl-GPC), but not the lysolecithin analogue 1-0-octadecyl 2-0-methylglycero phosphocholine, lowered phospholipase A2 activity with either labelling method. Dihexadecyl-GPC had an inhibitory effect on the release of arachidonic acid and eicosanoids. Moreover, this effect, as measured by tritiated nonesterified fatty acid formation, was greater in activated cells labelled with tritiated phospholipid (IC50 6 microM) than with [3H] arachidonic acid (IC50 60 microM). This is attributable to the inhibitory effect of dihexadecyl-GPC on endogenous phospholipase A2 and the endogenous enzyme excreted together with lysosomes into the medium. It may be concluded that radioactive phospholipid labelling is a sensitive method for measuring phospholipase A2 activity and assessing the effects of potential phospholipase inhibitors.