Multinucleated variant endothelial cells (MVECs) have frequently been observed in the human aorta, and the ratio of MVECs to typical endothelial cells correlates well with the severity of atherosclerosis. MVECs showed no capacity of proliferation in vitro, making their study extremely difficult. We attempted to obtain reproducible MVECs in vitro in order to understand their functional characteristics and their roles in atherosclerosis. This study was designed to derive MVECs from human umbilical cord vein endothelial cells (HUVECs) with different reagents such as Meso-4,4'-(2,3-butanediyl) bis (2,6-piperazinedione) (ICRF-193), low density lipoprotein (LDL), interleukin-4 (IL-4), polyethylene glycol (PG), H2O2, and linoleic acid hydroperoxide (LAHO). We found that 10 microM ICRF-193 was most effective in inducing MVECs. We then investigated the features of aortic endothelial cells (AECs) and ICRF-193 treated HUVECs (I-HUVECs) in the following four aspects: morphology, by light microscopy; cell cycle phase, by uptake of BrdU; expression of endothelial cell (EC) related markers such as von Willebrand Factor (vWF), endothelin-1 (ET-1), prostacyclin (PGI2) and intercellular adhesion molecule CD34 by immunocytochemistry; and biological activity by analyzing their uptake of low density lipoprotein (LDL). Furthermore, we compared aortic MVECs (AMVECs) and other aortic endothelial cells (A-others), as well as A-MVECs and ICRF-193 induced MVECs (I-MVECs) in every parameter examined. We found: 1. Compared with A-others, A-MVECs expressed more vWF (p < 0.01), more ET-1 (p < 0.05) and less CD34 (p < 0.01). In the uptake of LDL, A-MVECs took up more nLDL than A-others; 2. Both A-MVECs and I-MVECs contained multiple nuclei, but the nuclei differed in shape. A-MVECs and I-MVECs were similar in the nuclear incorporation of BrdU, in the uptake of nLDL and oxLDL, and in the expression of vWF, ET-1 and PGI2, but different in the expression of CD34 (p < 0.01). Our findings suggested that A-MVECs may transfer more plasma LDL to the subendothelial space because they took up more LDLs. I-MVECs were similar to A-MVECs morphologically and functionally. Thus, I-MVECs could be considered as substitutes in the study of A-MVECs.