A saturable blood-to-brain transport system for leptin across the blood-brain barrier (BBB) has been observed in vivo. Since the main component of the non-fenestrated microvessels of the BBB is the endothelial cell, we established an in vitro culture system of these cerebrovascular cells to study leptin transport and to determine whether the self-inhibition of leptin transport characteristic of a saturable system occurs at this level. The results show that 125I-leptin crossed from the luminal to abluminal side of a monolayer of cerebral microvessel cells significantly faster than the albumin and lactalbumin controls. This transport of 125I-leptin across an in vitro BBB was significantly faster than in the opposite direction and was dose-relatedly inhibited by the addition of unlabeled leptin. Thus, the results establish that the saturable transport system for leptin across the BBB occurs at the level of the endothelial cells of the BBB.