Serum leptin levels are upregulated in proportion to body fat and also increase over the short-term in response to meals or insulin. To understand the mechanisms involved, we assessed leptin synthesis and secretion in samples of adipose tissue from subjects with a wide range of body mass index (BMI). Tissue leptin content and relative rates of leptin biosynthesis as determined by metabolic labeling were highly correlated with each other, and with BMI, fat cell size. To understand mechanisms regulating leptin synthesis in obesity, we used biosynthetic labeling to directly assess the effects of insulin and glucocorticoids (dexamethasone) on leptin synthesis and secretion in human adipose tissue. Chronic treatment (1-2 days in organ culture) with insulin increased relative rates of leptin biosynthesis without affecting leptin mRNA levels. In contrast, dexamethasone increased leptin mRNA and biosynthesis in parallel. Acute treatment with insulin or dexamethasone (added during 1 h pre-incubation and 45 min pulse-labeling) did not affect relative rates of leptin biosynthesis, but pulse-chase studies showed that addition of insulin nearly doubled the release of ³⁵S-leptin after 1 h chase. We conclude that the higher leptin stores in adipose tissue of obese humans are maintained by chronic effects of insulin and glucocorticoids acting at pre- and post-translational levels, and that the ability of insulin to increase the release of preformed leptin may contribute to short-term variations in circulating leptin levels.