Platelet membranes contain two distinct transacylase activities catalyzing the synthesis of arachidonoyl phosphatides by acylation of added lysophosphatides with endogenous esterified arachidonate. In the absence of CoA, arachidonate is incorporated only into ethanolamine lysophosphatides with a high preference for the plasmalogen form (Kramer, R. M., and Deykin, D. (1983) J. Biol. Chem. 258, 13806-13811). In the presence of CoA, however, lysophospholipids are acylated in the order 1-acyl-lysophosphatidylserine greater than 1-acyl-lysophosphatidylethanolamine greater than 1-acyl-lysophosphatidylinositol. The CoA-mediated transacylation reaction was characterized with 1-acyl-lysophosphatidylserine as acyl acceptor. It was highly specific for arachidonate and preferentially used phosphatidylcholine as the arachidonoyl donor. This enzymatic pathway may be part of a deacylation-transacylation cycle for remodeling of phospholipids synthesized de novo (according to the Lands pathway) representing a mechanism for enrichment of phospholipids with arachidonic acid.