We investigated the in vitro regulation of leptin expression in adipose tissue from severely obese women and men before and after culture with insulin (7 nM) and/or dexamethasone (25 nM). Leptin mRNA and leptin secretion were two- to threefold higher in subcutaneous vs. omental adipose tissue before culture. Dexamethasone transiently increased leptin mRNA approximately twofold in both depots after 1 day of culture [P < 0.01 vs. basal (no hormone control)], but leptin secretion was only increased in omental adipose tissue (P < 0.005 vs. basal). Insulin did not increase leptin mRNA in either depot but increased leptin secretion approximately 1.5- to 3-fold in subcutaneous tissue throughout 7 days of culture (P < 0.05 vs. basal). The combination of insulin and dexamethasone increased leptin mRNA and leptin secretion approximately two- to threefold in both depots at day 1 (P < 0.005 vs. basal or insulin) and maintained leptin expression throughout 7 days of culture. We conclude that insulin and glucocorticoid have depot-specific effects and function synergistically as long-term regulators of leptin expression in omental and subcutaneous adipose tissue from obese subjects.