c-kit ligand (KL) activated mouse bone marrow-derived mast cells (BMMC) for the dose- and time-dependent release of arachidonic acid from cell membrane phospholipids, with generation of leukotriene (LT) C4 in preference to prostaglandin (PG)D2. KL at concentrations of 10 ng/ml elicited half-maximal eicosanoid generation and at concentrations of > 50 ng/ml elicited a maximal generation of approximately 15 ng LTC4 and 1 ng PGD2 per 10(6) cells, with 20% net beta-hexosaminidase release 10 min after stimulation. Of the other cytokines tested, none, either alone or in combination with KL, elicited or modulated the immediate phase of mediator release by BMMC, indicating strict specificity for KL. Activation of BMMC in response to KL was accompanied by transient phosphorylation of cytosolic phospholipase A2 and reversible translocation of 5-lipoxygenase to a cell membrane fraction 2-5 min after stimulation, when the rate of arachidonic acid release and LTC4 production were maximal. BMMC continuously exposed to KL in the presence of IL-10 and IL-1 beta generated LTC4 in marked preference to PGD2 over the first 10 min followed by delayed generation of PGD2 with no LTC4 over several hours. Pharmacologic studies revealed that PGD2 generation in the immediate phase depended on prostaglandin endoperoxide synthase (PGHS)-1 and in the delayed phase on PGHS-2. Thus, KL provided a nonallergic stimulus for biphasic eicosanoid generation by mast cells. The immediate phase is dominated by LTC4 generation with kinetics and postreceptor biosynthetic events similar to those observed after cell activation through the high affinity IgE receptor, whereas the delayed phase of slow and selective PGD2 production is mediated by induction of PGHS-2.