We have reported previously that creatine is biosynthesized from glycine and L-arginine in Müller cells, but the mechanism responsible for glycine and L-arginine uptake by Müller cells remains elusive. To explore this issue, [(14)C]glycine and [(3)H]L-arginine uptake by Müller cells was characterized using a conditionally immortalized rat Müller cell line (TR-MUL5 cells). [(14)C]Glycine uptake by TR-MUL5 cells was Na(+)- and Cl(-)-dependent, and a saturable process with Michaelis-Menten constants of 48.6 microM and 4.53 mM, and inhibited by glycine transporter 1 (GlyT1) and system A substrates. Under Cl(-)-free conditions, the high-affinity process was abolished. RT-PCR analysis demonstrated that GlyT1 and system A (encoding ATA1 and ATA2) mRNA are expressed in TR-MUL5 cells. These uptake studies suggest that GlyT1 and system A are involved in [(14)C]glycine uptake for the high- and low-affinity processes, respectively, in TR-MUL5 cells. [(3)H]l-Arginine uptake by TR-MUL5 cells was primarily an Na(+)-independent and saturable process with Michaelis-Menten constants of 15.0 and 403 microM. This process was inhibited by substrates of cationic amino acid transporter (CAT)s, such as L-arginine, L-lysine, and L-ornithine. The expression of CAT1 protein was detected in TR-MUL5 cells. These results suggest that CAT1 is at least involved in [(3)H]l-arginine uptake by TR-MUL5 cells. In conclusion, GlyT1 and CAT1 most likely mediate glycine and L-arginine uptake, respectively, by Müller cells and are expected to play an important role in supplying precursors for creatine biosynthesis in Müller cells.