Glycine is a major inhibitory neurotransmitter in the mammalian CNS (central nervous system). Glycinergic neurotransmission is terminated by the uptake of glycine into glycinergic nerve terminals and neighbouring glial cells. This uptake process is mediated by specific Na(+)/Cl(-)-dependent GlyTs (glycine transporters), GlyT1 and GlyT2. GlyT1, in addition, is thought to regulate the concentration of glycine at excitatory synapses containing NMDARs (N-methyl-D-aspartate receptors), which require glycine as a co-agonist. We have analysed the physiological roles and regulation of GlyT1 and GlyT2 by generating transporter-deficient mice and searching for interacting proteins. Our genetic results indicate that at glycinergic synapses, the glial transporter GlyT1 catalyses the removal of glycine from the synaptic cleft, whereas GlyT2 is required for the re-uptake of glycine into nerve terminals, thereby allowing for neurotransmitter reloading of synaptic vesicles. Both GlyT1 and GlyT2 are essential for CNS function, as revealed by the lethal phenotypes of the respective knockout mice. Mice expressing only a single GlyT1 allele are phenotypically normal but may have enhanced NMDAR function. GlyT2 is highly enriched at glycinergic nerve terminals, and Ca(2+)-triggered exocytosis and internalization are thought to regulate GlyT2 numbers in the pre-synaptic plasma membrane. We have identified different interacting proteins that may play a role in GlyT2 trafficking and/or pre-synaptic localization.