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d-serine, an endogenous co-agonist of NMDA receptors in vertebrate retina, may modulate glutamate sensitivity of retinal neurons. This study determined at the functional and molecular level the transport process responsible for d-serine in retinal Müller cells. RT-PCR and immunoblotting showed that serine racemase (SR), the synthesizing enzyme for d-serine, is expressed in the rMC-1 Müller cell line and primary cultures of mouse Müller cells (1°MCs). The relative contributions of different amino acid transport systems to d-serine uptake were determined based on differential substrate specificities and ion dependencies. d-serine uptake was obligatorily dependent on Na+, eliminating Na+-independent transporters (asc-1 and system L) for d-serine in Müller cells. The Na+:substrate stoichiometry for the transport process was 1:1. d-serine transport was inhibited by alanine, serine, cysteine, glutamine, and asparagine, but not anionic amino acids or cationic amino acids, suggesting that d-serine transport in Müller cells occurs via ASCT2 rather than ASCT1 or ATB0,+. The expression of mRNAs specific for ASCT1, ASCT2, and ATB0,+ was analyzed by RT-PCR confirming the expression of ASCT2 (and ASCT1) mRNA, but not ATB0,+, in Müller cells. Immunoblotting detected ASCT2 in neural retina and in 1°MCs; immunohistochemistry confirmed these data in retinal sections and in cultures of 1°MCs. The efflux of d-serine via ASCT2 by ASCT2 substrates was demonstrable using the Xenopus laevis oocyte heterologous expression system. These data provide the first molecular evidence for SR and ASCT2 expression in a Müller cell line and in 1°MCs and suggest that d-serine, synthesized in Müller cells by SR, is effluxed via ASCT2 to regulate NMDA receptors in adjacent neurons.