The subcellular localization of glycine transporters one (GLYT1) and two (GLYT2) stably expressed in PC12 cells has been studied. To facilitate visualization, enhanced green fluorescent protein (GFP) was fused to the amino terminus of both glycine transporters. Functional analysis of the GFP–GLYT1 and GFP–GLYT2 stable cell lines demonstrated that they exhibited high affinity for glycine and the characteristic properties of both glycine transporter subtypes. The GFP-coupled transporters were differently distributed throughout the cell. GFP–GLYT1 was mainly localized on the plasma membrane, whereas most of GFP–GLYT2 was present on large dense-core vesicles and endosomes. Both transporters were absent from the synaptic vesicle population in PC12 cells.