Objective— We previously reported that homocysteine (Hcy) inhibits endothelial cell (EC) growth and promotes vascular smooth muscle cell (VSMC) proliferation. This study characterized and directly compared Hcy transport in cultured human aortic ECs (HAECs) and smooth muscle cells (HASMCs). Methods and Results— Hcy (10 μmol/L) was transported into both cell types in a time-dependent fashion but was approximately 4-fold greater in HASMCs, and is nonstereoenantiomer specific. Hcy transport in HAECs had a Michaelis-Menten constant ( Km ) of 39 μmol/L and a maximal transport velocity ( Vmax ) of 873 pmol/mg protein/min. In contrast, Hcy transport in HASMCs had a lower affinity ( Km =106 μmol/L) but a higher transport capacity ( Vmax =4192 pmol/mg protein/min). Competition studies revealed that the small neutral amino acids tyrosine, cysteine, glycine, serine, alanine, methionine, and leucine inhibited Hcy uptake in both cell types, but the inhibition was greater for tyrosine, serine, glycine, and alanine in HAECs. Sodium-depletion reduced Hcy transport to 16% in HAECs and 56% in HASMCs. Increases in pH from 6.5 to 8.2 or lysosomal inhibitors blocked Hcy uptake only in HAECs. In addition, Hcy shares carrier systems with cysteine, in a preferable order of alanine-serine-cysteine (ASC)>aspartate and glutamate (X _AG )=large branched-chain neutral amino acids (L) transporter systems in HAECs and ASC>L>X _AG in HASMCs. The sodium-dependent system ASC plays a predominant role for Hcy transport in vascular cells. Conclusions— Transport system ASC predominantly mediates Hcy transport in EC and is lysosomal dependent.