Eicosapentaenoic acid (EPA), one of the n-3 polyunsaturated fatty acids, has been shown to stimulate leptin mRNA expression and secretion in 3T3-L1 cells. However, other studies have reported inhibitory effects of EPA on leptin expression and secretion in vivo and in vitro. In order to determine the direct effects of EPA on basal and insulin-stimulated leptin secretion, isolated rat adipocytes were incubated with EPA in the absence and presence of insulin. EPA (10, 100 and 200 µM) increased basal leptin gene expression and secretion (+43.8%, P < 0.05, +71.1%, P < 0.01 and +73.7%, P < 0.01, respectively). EPA also increased leptin secretion in the presence of 1.6 nM insulin, however the effect was less pronounced than in the absence of it. Because adipocyte glucose and lipid metabolism are involved in the regulation of leptin production, the metabolic effects of this fatty acid were also examined. EPA (200 µM) increased basal glucose uptake in isolated adipocytes (+50%, P < 0.05). Anaerobic metabolism of glucose, as assessed by lactate production and proportion of glucose metabolized to lactate, has been shown to be inversely correlated to leptin secretion and was decreased by EPA both in absence and in presence of insulin. EPA increased basal glucose oxidation as determined by the proportion of ¹⁴C-labeled glucose metabolized to CO₂. Lipogenesis (¹⁴C-labeled glucose incorporation into triglyceride) was decreased by EPA in absence of insulin, whereas lipolysis (glycerol release) was unaffected. The EPA-induced increase of basal leptin secretion was highly correlated with increased glucose utilization (r = +0.89, P < 0.01), and inversely related to the anaerobic glucose metabolism to lactate. EPA's effect on insulin-stimulated leptin secretion was not related to increased glucose utilization, but was inversely correlated with anaerobic glucose metabolism to lactate (r = -0.84, P < 0.01). Together, the results suggest that EPA, like insulin, stimulates leptin production by increasing the non-anaerobic/oxidative metabolism of glucose.