5,12-Dihydroxy-6,8,10,14-eicosatetraenoic acid was prepared from rabbit neutrophils challenged with ionophore A23187 plus arachidonic acid. It was purified by reverse-phase high-performance liquid chromatography with the use of a two-step procedure that effectively resolved the lipid from closely eluting contaminants. The phospholipid 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine was prepared from beef heart plasmalogen. Confirming earlier reports, both lipids stimulated human polymorphonuclear neutrophils to release granule-bound enzymes. Their respective degranulating actions took less than 5 minutes to become maximal, were dependent upon the presence of cytochalasin B, and required intact pathways of arachidonic acid metabolism, as judged by the inhibitory actions of arachidonate antimetabolites. Neither lipid required more than trace concentrations of extracellular calcium to effect optimal amounts of enzyme release. In one or more of these regards the two lipids differed strikingly from other neutrophil-degranulating agents such as A23187, N-formyl-methionyl-leucyl-phenylalanine, and phorbol myristate acetate. The mechanism of action of the two lipid stimuli, therefore, appear to differ somewhat from the mechanisms of the other three stimuli.