Leptin, the ob gene product, is produced by adipocytes, and it acts to decrease caloric intake and increase energy expenditure. To better understand the molecular mechanisms of hormone-regulated leptin synthesis and secretion, we assessed the ability of insulin and dexamethasone to acutely modulate ob gene expression and leptin secretion in rat adipocytes. Incubation of rat adipocytes with 100 nmol/l insulin for 2 h had no effect on ob mRNA levels, but it stimulated a twofold increase in leptin secretion. Dexamethasone (100 nmol/l) stimulated both a two- to fourfold increase in ob mRNA and a twofold increase in leptin secretion. Consonant with a posttranscriptional and transcriptional regulatory mechanism for insulin- and dexamethasone-stimulated leptin secretion, respectively, actinomycin D blocked dexamethasone-stimulated leptin secretion but did not affect insulin-stimulated leptin secretion. Cycloheximide treatment did not significantly affect ob mRNA accumulation, but it reduced total secreted leptin. Interestingly, however, insulin was still able to stimulate a twofold increase in leptin secretion. These data suggest that insulin, but not dexamethasone, is able to stimulate leptin secretion from a preexisting intracellular pool, although de novo protein synthesis is required for the full insulin-stimulated effect. Signaling pathways involved in leptin synthesis/secretion were also evaluated. The phosphatidylinositol 3-kinase inhibitor LY294002, the Map/Erk kinase inhibitor PD98059, and the immunosuppressant rapamycin had no effect on basal levels of leptin secretion. However, all three inhibitors markedly decreased both insulin- and dexamethasone-stimulated leptin secretion. These findings suggest a complex set of signaling pathways involved in mediating insulin- and dexamethasone-stimulated leptin synthesis and secretion.