Suspensions of human peripheral blood leukocytes were incubated with arachidonic acid and calcium ionophore A23187. Acidic lipid extracts were purified by silicic acid chromatography and reversed phase high performance liquid chromatography. A polar metabolite with an ultraviolet absorption spectrum identical to that of leukotriene B4 was identified by gas chromatography and mass spectrometry as a 5,12,20-trihydroxy-6,8,10,14-eicosatetraenoic acid. The data indicated that this compound was the 20-hydroxy metabolite of leukotriene B4. The compound showed chemokinetic activity towards human polymorphonuclear leukocytes and aggregating activity towards rat peritoneal polymorphonuclear leukocytes. Its activity was greater than that of 5-hydroxy-6,8,11,14-eicosatetraenoic acid but less than that of leukotriene B4. It is concluded that the 15,12,20-trihydroxyeicosatetraenoic acid may play a role as a mediator of inflammatory reactions.