Lipoprotein lipase (LPL) is the rate-limiting enzyme in the hydrolysis of serum triglycerides associated with the lipoprotein particles very low density lipoprotein and chylomicrons. The cell biology of LPL is complex. It functions while tethered to the extracellular matrix of the capillary endothelium. LPL is synthesized, however, in the parenchymal cells (for example, adipocytes or muscle cells) subtending the endothelium. Thus, after synthesis in and release by the parenchymal cell, LPL must move to the endothelial cell and across this cell monolayer before expression at its physiologically relevant location. LPL expression on the endothelium is regulated by insulin. The intent of this study was to ascertain the role of microvessel endothelial cells in the release of LPL from 3T3-L1 adipocytes and to ascertain whether insulin regulates the function of the endothelial cells. Endothelial cells were treated with insulin, and the resultant culture medium conditioned by the endothelial cells was placed on 3T3-L1 adipocytes. The release of LPL from the adipocytes induced by the endothelial cell-conditioned medium was then quantitated. Insulin concentrations as low as 100 pm stimulated the release of a factor from the endothelial cells. This factor, when added to adipocytes, caused the quantitative release of LPL from the plasma membrane of the adipocytes. The effect of insulin on the endothelial cells was maximal within 15 min of insulin addition to the endothelial cells. Repeated challenges of the endothelial cells with insulin resulted in the repeated release of the LPL release factor from the endothelial cells if the challenges were separated by periods of 2–3 h. However, if the endothelial cells were chronically stimulated with insulin for 18 h, a subsequent acute stimulation with insulin did not generate any LPL release factor. Thus, microvessel endothelial cells regulate the mobilization of LPL from adipocytes in an insulin-dependent manner.