The lack of the glycine transporter 1 (GlyT1) leads to early postnatal death due to failure of respiratory network activity. Here we demonstrate a segregated expression of GlyT1 on different astroglial cell populations of the ventral respiratory group. In TgN(hGFAP-EGFP) mice a combined immunohistochemical and electrophysiological approach was used to define the cellular expression of GlyT1 in the respiratory network. EGFP-labeled cells with outwardly rectifying current–voltage relationship did not express glycine transporter 1, while GlyT1 was abundantly expressed in mature protoplasmic astrocytes, which are electrophysiologically characterized by a large potassium conductance, a more negative membrane potential and the expression of glutamate transporters. Taken together, the vital capacity for the clearance of extracellular glycine is restricted to a subpopulation of astroglial cells.