Glycine transporter 2 (GLYT2) is specifically localized on the membrane in glycinergic nerve terminals and plays a critical role in retrieval of glycine from synaptic cleft for refilling the synaptic vesicles with it. Therefore, GLYT2 is a useful marker of glycinergic neuron. GLYT2 promoter-driven EGFP or Cre-expressing mouse lines, which are bacterial artificial chromosome (BAC) transgenic ones, were generated (Zeihofer et al., 2005; Ishihara et al., 2010). Because BAC vectors can contain large fragment of DNA, BAC transgenic animals are useful for expressing the target gene in the target neurons. However, knock-in method is superior to BAC transgenic one in expression reproducibility of the target gene. In order to realize specific gene ablation or expression in glycinergic neurons, we generated a GLYT2-Cre knock-in mouse line. The spatial activities of Cre were examined by crossing GLYT2-Cre mice with two kinds of Cre reporter mouse lines, ROSA26R and CAG-CAT-EGFP, which express β-gal or EGFP depending on Cre activity. The Cre activity was primarily distributed in the brainstem, cerebellum and spinal cord. In the brainstem, Cre activity was strongly detected in the medial vestibular nucleus, solitary nucleus, nucleus of the trapezoid body, and gigantocellular reticular nucleus. Moreover, Cre activity was also distributed in the cerebellar cortex, deep cerebellar nucleus, and gray matter of spinal cord. These areas are consistent with those in which GLYT2 mRNAs were present. In order to confirm that GLYT2-Cre activities were localized to glycinergic neurons, we are performing double immunohistochemical staining and/or double in situ hybridization analysis.