Lipoprotein lipase (LPL) that is associated with the luminal surface of capillary endothelial cells hydrolyzes circulating lipoprotein triglyceride molecules. Because LPL is synthesized by cells on the abluminal side of endothelial cells, LPL must contact both the abluminal as well as the luminal sides of the endothelium. To determine whether LPL interacts identically with apical (luminal) and basolateral (abluminal) sides of endothelial cells, we investigated binding, transport, and cellular uptake of LPL presented to each side of bovine aortic endothelial cell monolayers grown on semipermeable filters. When LPL was included in the medium on either the apical or basolateral side of the cells, a similar amount of LPL was found in the medium on the opposite side of the cells. Heat-inactivated LPL crossed the monolayers more rapidly in both directions. When cell surface LPL was assessed, more LPL bound to the apical than the basolateral endothelial cell surface. Release of cell surface-associated LPL was assessed with the use of heparin. Less heparin was required to dissociate apical-surface LPL. When LPL (4 micrograms/ml) was in contact with the apical surface for 1 hour, 32.8 +/- 4.9 ng LPL per 24-mm filter were internalized by the cells. If the LPL was in the basolateral medium, only 6 +/- 1.8 ng LPL were found inside the cells. Heat inactivation decreased LPL binding to cell surfaces and internalization by the cells. LPL interactions with the cells were also studied morphologically by using Texas Red (TR)-labeled LPL and confocal microscopy. More TR-LPL was associated with and internalized by the apical endothelial surface. Incubation of cells with TR-LPL in the basolateral medium led to accumulation of LPL on the apical surface, suggesting that the LPL was transported across the cells. Inclusion of TR-LPL on the apical surface did not lead to appreciable accumulation of LPL on the basolateral cell surface. Therefore, endothelial cells are polarized to accumulate LPL on the apical surface. In addition, more LPL is internalized from this side of the cells. We postulate that the polarity of endothelial cells allows LPL to collect at its physiological site of action, i.e., on the luminal surface.