We report here on the characterization of a mouse N-system amino acid transporter protein, which is involved in the transport of glutamine. This protein of 485 amino acids shares 52% sequence homology with an N-system amino acid transporter, mouse N-system amino acid transporter (mNAT) and its orthologs. Because this protein shares a high degree of sequence homology and functional similarity to mNAT, we named it mNAT2. mNAT2 is predominately expressed in the retina and to a slightly lesser extent in the brain. In the retina, it is located in the axons of ganglion cells in the nerve fiber layer and in the bundles of the optic nerve. Functional analysis of mNAT2 expressed in Xenopus oocytes revealed that the strongest transport activities were specific for l-glutamine. In addition, mNAT2 is a Na(+)- and pH-dependent, high affinity transporter and partially tolerates substitution of Na(+) by Li(+). Additionally, mNAT2 functions as a carrier-mediated transporter that facilitates efflux. The unique expression pattern and selective glutamine transport properties of mNAT2 suggest that it plays a specific role in the uptake of glutamine involved in the generation of the neurotransmitter glutamate in retina.