Leukotriene B 4 (LTB 4 ) is a potent lipid mediator involved in host defense and inflammatory responses. It causes chemotaxis, generation of reactive oxygen species, and degranulation. However, only little is known of the molecular mechanisms by which LTB 4 induces these biological activities. To analyze the intracellular signaling pathways to mediate lysosomal enzyme release through the cloned LTB 4 receptor (BLT1), we transfected BLT1 to rat basophilic leukemia cells (RBL-2H3). LTB 4 dose-dependently released -hexosaminidase, and the release was mostly inhibited when the cells were pretreated with pertussis toxin, indicating that the degranulation is mediated by G i proteins. LTB 4 activated phosphatidylinositol 3-kinase (PI3-K) through G i , and inhibition of PI3-K by wortmannin or LY290042 inhibited degranulation. Granulocytes from PI3-K␥-deficient mice showed reduced LTB 4 -induced degranulation, suggesting that this isozyme of PI3-K is involved in the degranulation. LTB 4 also caused calcium release from intracellular stores and calcium influx from the outside milieu through G i , but only the calcium influx is critical for the lysosomal enzyme release. Calcium influx and PI3-K activation are both downstream events of G i , since they were inhibited by pertussis toxin. These two events are in essence independent each other, because calcium depletion did not affect PI3-K, and inhibition of PI3-K did not attenuate calcium influx significantly. Thus, our results have clearly shown that LTB 4 binds BLT1 and activates G i -like protein, and both PI3-K␥ activation and a sustained calcium elevation by calcium influx are necessary for enzyme release in these cells.Leukotreine B 4 (LTB 4 ), 1 a metabolite of arachidonic acid, is one of the most potent chemoattractants for neutrophils. LTB 4 activates leukocytes, leading to chemotaxis, release of lysosomal enzyme, and production of superoxide anions, thus playing important roles in host defense (1, 2). Overproduction of LTB 4 is involved in various inflammatory diseases including psoriasis (3), bronchial asthma (4), rheumatoid arthritis (5), inflammatory bowel diseases (6), and ischemic renal failure (7). LTB 4 binds to a specific G protein-coupled receptor named BLT1, which was cloned in our laboratory (8). Signaling to activate leukocytes through BLT1 is poorly understood, in part because most of the previous studies have been done using peripheral leukocytes, especially granulocytes. The lifetime of these cells is short (several days), which makes it difficult to perform studies using various drugs or gene transfection. We have previously analyzed signaling pathway from BLT1 using Chinese hamster ovary cells stably expressing human BLT1 (8). In these cells, LTB 4 increases intracellular IP3 levels by G q -like protein(s), inhibits adenylyl cyclase activity, and activates chemotaxis through G i -like protein(s). In hematopoietic cells, however, the intracellular events leading to release of lysosomal enzymes and generation of oxygen species are poorly understood....