Neurotransmitter transport systems are major targets for therapeutic alterations in synaptic function. We have cloned and sequenced a cDNA encoding the human type 2 glycine transporter GlyT2 from human brain and spinal cord. An open reading frame of 2391 nucleotides encodes a 797 amino acid protein that transports glycine in a Na+/Cl--dependent manner. When stably expressed in CHO cells, human GlyT2 displays a dose-dependent uptake of glycine with an apparent Km of 108 microM. This uptake is not affected by sarcosine at concentrations up to 1 mM. Radiation hybrid analysis mapped the GlyT2 gene to D11S1308 (LOD=8.988) on human chromosome 11p15.1-15.2.