D-Serine, a coagonist for N-methyl-D-aspartate-type glutamate receptors, which mediate visual signal transmission, is thought to be generated from L-serine via serine racemase in the retina. However, the source of L-serine and D-serine in the retina are yet to be determined. The purpose of the present study was to investigate the characteristics of the blood-to-retina transport of serine at the inner blood-retinal barrier (BRB). In vivo study revealed the bloodto-retina transport of [ 3 H]L-serine with an influx clearance of 49.9 :L/(min•g retina), which is greater than that of [ 3 H]D-serine. This was consistent with the L-isomer-predominant uptake of serine by conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), an in vitro inner BRB model. [ 3 H]L-Serine and [ 3 H]D-serine uptake by TR-iBRB2 cells took place in an Na + -dependent and a concentration-dependent manner with Michaelis constant values of 97.5 :M and 9.63 mM, respectively. The uptake process of [ 3 H]L-serine and [ 3 H]D-serine was significantly inhibited by system ASC (alanine-serine-cysteine) substrates. Polymerase chain reaction analysis and immunocytochemistry revealed the expression of ASC transporters ASCT1 and ASCT2 in TR-iBRB2 cells. These results suggest that the system ASC at the inner BRB is a potent pathway for supplying serine in the form of the L-isomer from the circulating blood to the retina.