Leptin is an adipocyte-derived hormone that is found in the peripheral circulation in correlation with total body fat mass. While leptin is best known for its ability to regulate energy homeostasis, it is a pleotropic hormone whose receptor is widely distributed in various tissues including cells of the immune system. We have previously reported that leukotriene (LT) synthesis in macrophages from leptin-deficient mice is attenuated and that the provision of exogenous leptin restores this defect. To explore the possibility that leptin regulates eicosanoid synthesis in cells from normal animals, we cultured rat alveolar and murine peritoneal macrophages overnight with media alone or with increasing doses of leptin. Following stimulation with calcium ionophore (A23187) or zymosan, we observed that leptin enhanced prostaglandin E₂ and LT synthesis in a dose-dependent manner. Since arachidonic acid (AA) is the metabolic precursor of the eicosanoids, we next asked whether leptin regulates AA release and phospholipase A₂ (PLA₂) activity. Rat alveolar macrophages (AMs) were prelabeled overnight with [³H]AA and cultured with increasing doses of leptin on the following day. Leptin enhanced A23187-stimulated [³H]AA release in a dose-dependent manner that peaked at 5 ng/ml (300% above control). Using rat AMs pretreated overnight with leptin, we observed that leptin enhanced both calcium-dependent and calcium-independent PLA₂ activity. To determine the time required for leptin to enhance AA release, AMs were prelabeled overnight with [³H]AA, cultured with leptin (5 ng/ml) on the following day, and cell culture medium was collected following stimulation with A23187. Leptin enhanced AA release at 30 min, and 1, 2, 4, and 16 hours following A23187 stimulation. To explore the mechanism by which short-term leptin pretreatment enhances AA release, we assessed cPLA₂α phosphorylation in rat AM lysates following leptin pretreatment. Western blot analysis of immunoprecipitated cPLA₂α revealed that leptin pretreatment of rat Ams increased cPLA₂α phosphorylation (ser505) in a dose-dependent manner that peaked at 15 min (sevenfold increase with 10 ng/ml) before returning to baseline levels at 60 min. To determine the mechanism by which long-term leptin pretreatment enhances macrophage AA release and LT synthesis, we assessed the expression of 5-lipoxygenase, 5-lipoxygenase activating protein, and cPLA₂α in murine peritoneal macrophages and of cPLA₂γ in rat AMs using western blot analysis. Overnight pretreatment with leptin increased the expression of 5-lipoxygenase (200%) in murine peritoneal macrophages and of cPLA₂γ (180%) in rat AMs. Our data suggest that the increased incidence of inflammatory diseases, such as asthma and osteoarthritis, associated with obesity may be related to the ability of leptin to increase AA release and eicosanoid synthesis.