In our previous report (Shikano, M., Masuzawa, Y. and Yazawa, K. (1993) J. Immunol. 150, 3525-3533), we described that the enrichment of docosahexaenoic acid (DHA, 22:6(n - 3)) reduces both arachidonic acid (AA, 20:4(n - 6)) release and platelet-activating factor (PAF) synthesis in human eosinophilic leukemia cells, Eol-1. Since no DHA release was observed in response to Ca-ionophore stimulation, we presumed that the phospholipase A2 (PLA2) responsible for AA release and PAF synthesis can not hydrolyze the DHA moiety of phospholipids. In the present paper, we examined whether DHA-containing diacyl- and alkenylacylglycerophosphoethanolamine (DHA-diacylGPE and DHA-alkenylacyGPE) are susceptible to the action of AA-preferential 85 kDa cytosolic phospholipase A2 (cPLA2) from rabbit platelets in comparison with AA and eicosapentaenoic acid (EPA, 20:5(n - 3)) derivatives. When diacylGPE was used as a substrate, DHA release was almost negligible under the assay condition that allowed AA and EPA to be liberated at the rates of 4.3 mumol/min per mg protein and 2.5 mumol/min per mg protein, respectively. On the other hand, 14 kDa type II PLA2 hydrolyzed DHA-diacylGPE as well as AA-diacylGPE and EPA-diacylGPE. When DHA-diacylGPE and AA-diacylGPE were mixed at equimolar concentrations, DHA release by cPLA2 was not observed and AA release was reduced to 32% in the case without DHA-diacylGPE. This indicated that DHA-diacylGPE is a poor substrate but possesses the inhibitory activity for cPLA2. cPLA2 does not clearly discriminate between AA-alkenylacylGPE and AA-diacylGPE. As in the case using diacylGPE as a substrate, DHA-alkenylacylGPE was completely discriminated from AA-alkenylacylGPE by cPLA2. The roles of DHA and cPLA2 in the synthesis of lipid mediators will be discussed in relation to the new aspects of the substrate specificity of cPLA2 provided here.