The mechanism by which calcium regulates leptin secretion was studied in adipocytes isolated from rat white adipose tissue. Incubation of adipocytes in a medium containing glucose, but no calcium, markedly inhibited insulin-stimulated leptin secretion (ISLS) and synthesis, without affecting basal leptin secretion or lipolysis. However, when pyruvate was used as a substrate, ISLS was insensitive to the absence of calcium. Likewise, the stimulatory effects of insulin were completely prevented by phloretin, cytochalasin B and W13 (3 agents that interfere with early steps of glucose metabolism) in the presence of glucose, but not in the presence of pyruvate. Thus, calcium appears to be specifically required for glucose utilization. On the other hand, ⁴⁵Ca uptake and leptin secretion were not affected by insulin or by inhibitors of L-type calcium channels. However, agents increasing plasma membrane permeability to calcium (high calcium concentrations, A23187 and ATP) increased ⁴⁵Ca uptake and concomitantly inhibited ISLS. Similarly, release of endogenous calcium stores by thapsigargin inhibited ISLS in a dose-dependent manner. ATP, A23187, calcium and thapsigargin inhibited ISLS even in the presence of pyruvate. These results show that 1) extracellular calcium is necessary for ISLS mainly by affecting glucose uptake, 2) insulin does not affect extracellular calcium uptake and 3) increasing cytosolic calcium by stimulating its uptake or its release from endogenous stores inhibits insulin-stimulated leptin secretion at a level independent of glucose metabolism. Thus, calcium regulates leptin secretion from adipocytes in a manner that is markedly different from its role in the exocytosis of many other polypeptidic hormones.