We have isolated a cDNA encoding a high affinity, Na+/Cl(-)-dependent glycine transporter, GLYT-2, which is distinct from another glycine transporter, GLYT-1. While the 3' sequences of these two cDNAs are identical, the 5' noncoding regions and the N-termini are completely different. GLYT-1 is found only in the white matter of the CNS, while GLYT-2 is found in the gray matter of the CNS as well as in macrophages and mast cells in peripheral tissues. Our findings suggest that tissue-specific alternative splicing or alternative promoter usage from a single gene results in two mRNA products encoding similar but distinct glycine transporters. The anatomic distribution of GLYT-2 mRNA supports the emerging status of glycine as a supraspinal neurotransmitter and suggests that glycine may function as a chemical messenger outside the CNS.