High density lipoproteins (HDL) and their main protein constituent, apolipoprotein A-I (apoA-I), exert potentially anti-atherogenic properties within the arterial wall. However, it is unknown how they are transported from the blood stream into the vascular wall. Here we investigated the interaction of apoA-I with endothelial cells. At 4 degrees C endothelial cells bound 125I-apoA-I with high affinity, Kd = 2.1 microg/ml and in a saturable manner (Bmax of 35 ng/mg cell protein). At 37 degrees C, the cell association of apoA-I revealed similar affinity as at 4 degrees C (Kd = 2.2 microg/ml) but the maximum specific cell association was much enhanced (Bmax = 360 ng/mg cell protein). Binding and cell association was competed by excess unlabeled apoA-I and HDL but not by albumin. Biotinylation experiments and electron microscopy studies showed that endothelial cells internalize labeled apoA-I. Only minor amounts of the internalized apoA-I were degraded. Cultivated in a Transwell system, the cells transported a fraction of 125I-apoA-I from the apical to the basolateral compartment in a competable and temperature-sensitive manner. Furthermore, after specific transport the originally prebeta-mobile and lipid-free apoA-I was recovered as particles which have electrophoretic alpha-mobility. We conclude that endothelial cells transcytose and lipidate lipid-free apoA-I.