Leptin, the peptide encoded by the obese gene, is secreted by adipose cells and plays a role in regulating food intake, energy expenditure, and adiposity. Because earlier studies suggested that insulin increases the expression of leptin, we investigated the effect of insulin on leptin secretion by adipose tissue. Epididymal fat pads were incubated in vitro in the presence or absence of insulin over a 4-h time course. Insulin increased leptin secretion by about 80% at all time points studied. After 10 min of insulin treatment, the amount of tissue-associated leptin was lower in insulin-stimulated tissue, presumably due to the increased secretion. At later times, both tissue-associated leptin and total leptin production were higher in insulin-treated tissue. In untreated, isolated adipose cells, immunostaining of leptin was detected in the endoplasmic reticulum by confocal microscopy. After insulin treatment, there were two populations of cells. In many cells, leptin staining became fainter and was restricted to a narrow band near the plasma membrane. However, in other cells the leptin-staining pattern was unchanged. Leptin did not colocalize with GLUT4, the glucose transporter isoform found primarily in insulin-responsive cells, in either basal or insulin-stimulated adipose cells. In this study, insulin increased both secretion and production of leptin by adipose tissue fragments. Interestingly, insulin appeared to stimulate the transport of leptin from the endoplasmic reticulum rather than acting on a pool of regulated secretory vesicles.