Leptin and melatonin play an important role in the regulation of body mass and energy balance. Both hormones show a circadian rhythm with increasing values at night. In addition, melatonin receptors were recently described in adipocytes, where leptin is synthesized. Here, we investigated the influence of melatonin and its interaction with insulin and dexametasone on leptin expression. Isolated rat adipocytes were incubated with melatonin (1nM) alone or in combination with insulin (5nM) and/or dexamethasone (7nM) for 6 h. Melatonin or insulin alone did not affect leptin expression, but together, they increased it by 120%. Dexamethasone increased leptin mRNA content (105%) and this effect was not enhanced by melatonin. The simultaneous treatment with the three hormones provoked a further increase in leptin release (250%) and leptin mRNA (100%). Melatonin prevented the forskolin-induced inhibition (95%) of leptin expression. In addition, melatonin's ability to stimulate leptin release (in the presence of insulin) was completely blocked by pertussis toxin and luzindole. To get further insight in the molecular basis of melatonin and insulin synergism, the insulin signaling pathway was investigated. Melatonin increased the insulin-induced insulin receptor- tyrosine phosphorylation which led to an increased serine phosphorylation of the downstream convergent protein AKT. We concluded that melatonin interacts with insulin and upregulates insulin-stimulated leptin expression. These effects are due to melatonin binding to the pertussis toxin-sensitive Gi-protein coupled membrane receptor (MT₁ subtype) and the cross-talk with insulin, since insulin receptor and its convergent target AKT are co-activated by melatonin.