The type-2 glycine transporter GLYT2 is expressed in glycinergic neurons and is involved in the termination of inhibitory neurotransmission at strychnine-sensitive glycinergic synapses. We isolated cDNA of a GLYT2 isoform, GLYT2a, from mouse brain, and found that it contains a coding sequence of 798 amino acids. We also isolated and characterized the mouse GLYT2 (mGLYT2) gene, which was found to be divided into 18 exons and spread over 55 kb. 5'-rapid amplification of cDNA ends analyses demonstrated the existence of another two isoforms, mGLYT2b and mGLYT2c, in addition to mGLYT2a. Both mGLYT2b and mGLYT2c would produce a protein eight amino acids shorter than mGLYT2a. Analysis of the genomic clones encompassing the 5'-exons revealed that the three transcripts arose from a single gene by alternative splicing. RT-PCR analysis indicated that all three mGLYT2 isoforms were expressed at high levels in brain stem and spinal cord. These data will be useful for investigating the function of GLYT2 proteins and glycinergic neurons by gene targeting experiments.