A 96-well plate format system is described for the in vitro culture and analysis of leptin secretion by mature adipocytes. Cultured adipocytes secreted leptin in a linear fashion over a 48 h period and secretion was inhibited by actinomycin D treatment. Dexamethasone and insulin stimulated leptin production in vitro, with dexamethasone proving a more potent stimulus throughout. Culture of adipocytes with insulin and dexamethasone together resulted in an additive release of leptin, suggesting that stimulation by these factors operates via independent routes. Isoprenaline (1 - 1000 microM) was a potent inhibitor of leptin production but propanolol (3 microM) could block this inhibition. Inclusion of growth hormone, insulin-like growth factor 1 or tumor necrosis factor alpha did not affect leptin secretion by mature adipocytes.