Na+/Cl(-)-dependent glycine transporters are crucial for the termination of neurotransmission at glycinergic synapses. Two different glycine transporter genes, GlyT1 and GlyT2, have been described. Several isoforms differing in their 5′ ends originate from the GlyT1 gene. We have determined the genomic structure of the murine GlyT1 gene to elucidate the genetic basis underlying the different isoforms. Analysis of cDNA 5′-ends revealed that the GlyT1a and 1b/1c mRNAs are transcribed from two different promoters. During murine embryonic development GlyT1 mRNAs were detectable by RNase protection assays as early as embryonic day E9 and reached maximal levels between E13 and E15. In situ hybridization revealed GlyT1 expression in the developing spinal cord mainly in the ventral part of the ventricular zone at E12. At later stages (E15) transcripts were also found in the lateral half of the basal and intermediate gray matter. In contrast, the second glycine transporter gene GlyT2 displayed a completely different expression pattern. At E11 it is expressed in the mantle zone, and at later stages throughout the ventral horns. In the adult rat brain and spinal cord, GlyT1 hybridization signals were found exclusively in glial cells. Our data indicate that GlyT1 is an early marker of neural development and encodes glia-specific transporter proteins.