We investigated the effects of high-density lipoprotein (HDL) on the intracellular pH (pH i), and on the proliferation of human vascular endothelial cells (HUVEC), as well as on their production of prostacyclin (PGI 2). The pH i was slightly acidified when extracellular Ca 2+ was chelated with EGTA. Pretreatment of HUVEC with amiloride, the Na +/H + exchange inhibitor, caused the pH i to become strongly acidic. The addition of HDL produced a biphasic shift in pH i, with a brief initial acidification followed by a rapid alkaline shift. The initial decrease in pH i was abolished in the cells pretreated with EGTA, and subsequent alkalinization was inhibited. The alkalinization of pH i disappeared in the cells pretreated with amiloride. These results suggest that pH i depends mainly on Na +/H + exchange and partially on the extracellular Ca 2+ of the HUVEC either in the resting unstimulated state or during HDL stimulation. In contrast, the addition of LDL produced an acidification of pH i, which was increased by LDL in the Ca 2+-free condition. In the cells pretreated with amiloride, pH i was not further acidified by LDL. As a result, HDL promoted the proliferation of cells, an action that was inhibited by pretreatment with EGTA. However LDL inhibited cell proliferation, an action unaffected by EGTA pretreatment. The addition of HDL also enhanced the generation of prostacyclin in endothelial cells, the enhancement of PGI 2 generation resulted from an increase in the release of Ca 2+ from storage sites, due not only to an increased production of inositol 1,4,5-trisphosphate (IP 3), but also to the alkalinization of pH i. These effects may be involved in the mechanism of HDL's anti-atherosclerotic action.