Although group VIA Ca 2؉ -independent phospholipase A 2 ␤ (iPLA 2 ␤) has been implicated in various cellular events, the functions of other iPLA 2 isozymes remain largely elusive. In this study, we examined the cellular functions of group VIB iPLA 2 ␥. Lentiviral transfection of iPLA 2 ␥ into HEK293 cells resulted in marked increases in spontaneous, stimulus-coupled, and cell deathassociated release of arachidonic acid (AA), which was converted to prostaglandin E 2 with preferred cyclooxygenase (COX)-1 coupling. Conversely, treatment of HEK293 cells with iPLA 2 ␥ small interfering RNA significantly reduced AA release, indicating the participation of endogenous iPLA 2 ␥. iPLA 2 ␥ protein appeared in multiple sizes according to cell types, and a 63-kDa form was localized mainly in peroxisomes. Electrospray ionization mass spectrometry of cellular phospholipids revealed that iPLA 2 ␥ and other intracellular PLA 2 enzymes acted on different phospholipid subclasses. Transfection of iPLA 2 ␥ into HCA-7 cells also led to increased AA release and prostaglandin E 2 synthesis via both COX-1 and COX-2, with a concomitant increase in cell growth. Immunohistochemistry of human colorectal cancer tissues showed elevated expression of iPLA 2 ␥ in adenocarcinoma cells. These results collectively suggest distinct roles for iPLA 2 ␤ and iPLA 2 ␥ in cellular homeostasis and signaling, a functional link between peroxisomal AA release and eicosanoid generation, and a potential contribution of iPLA 2 ␥ to tumorigenesis.Phospholipase A 2 (PLA 2 ) 1 catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to yield free fatty acids, including arachidonic acid (AA), and lysophospholipids. Mammalian PLA 2 enzymes have been classified into four major families, including secretory PLA 2 , cytosolic PLA 2 (cPLA 2 ), Ca 2ϩ -independent PLA 2 (iPLA 2 ), and platelet-activating factor acetyl hydrolases, each of which occurs as multiple isoforms (1, 2). Of these, the group IV cPLA 2 and group VI iPLA 2 families represent intracellular enzymes with a catalytic serine in their lipase consensus motif. Of the cPLA 2 family, cPLA 2 ␣, cPLA 2 ␤, and cPLA 2 ␦ possess a Ca 2ϩ -regulated C2 domain near the N terminus, and their function is Ca 2ϩ -dependent, whereas cPLA 2 ␥ is Ca 2ϩ -independent and membranebound (3-6). The regulatory roles for cPLA 2 ␣ in production of lipid mediators, including prostaglandins and leukotrienes, have been well documented in many studies, including gene targeting (7-10).Of the emerging iPLA 2 family, group VIA iPLA 2 ␤ has been implicated in various cellular events such as the basal fatty acid reacylation reaction (membrane remodeling) (11, 12), agonist-stimulated AA release and eicosanoid generation (13-16), cell proliferation (17), apoptosis (18 -21), exocytosis (22, 23), vascular relaxation (24), adipogenesis (25), and activation of store-operated channels and capacitative Ca 2ϩ influx (26). In iPLA 2 ␤ transgenic mice, the enzyme is activated during myocardial infarction and causes malignant ventr...