An analogue of arachidonic acid in which the COOH group is replaced by a trifluoromethyl ketone group (COCF3) has recently been shown to be a tight binding inhibitor of the 85-kDa cytosolic phospholipase A2 that is found in platelets and other cells (Street, I. P., Lin, H.-K., Laliberté, F., Ghomashchi, F. G., Wang, Z., Perrier, H., Tremblay, N. M., Huang, Z., Weech, P. K., and Gelb, M. H. (1993) Biochemistry 32, 5935-5940). This trifluoromethyl ketone inhibits most of the arachidonate release from the phospholipid pool in thrombin-stimulated human platelets at concentrations of 0-40 microM with 4 x 10(8) platelets/ml. A structure-function analysis of related compounds reveals a good correlation between the inhibition of the purified phospholipase A2 and the blockage of arachidonate release in platelets. A number of recently described potent inhibitors of the 14-kDa phospholipase A2 that is secreted from activated platelets have no effect on the level of free arachidonate production. Furthermore, the addition of a large amount of recombinant 14-kDa phospholipase A2 to platelets does not produce free arachidonate, and it does not alter the amount of arachidonate released following platelet activation with thrombin. These studies provide strong pharmacological evidence for the role of the cytosolic phospholipase A2 in producing most, if not all, of the liberated arachidonate in thrombin-stimulated human platelets, and they show that tight binding membrane-residing inhibitors of the cytosolic phospholipase A2 can block the eicosanoid cascade in living cells.