The glycine transporter GLYT2 is present in neurons of the spinal cord, the brain stem and the cerebellum. This localization is similar to that of glycine immunoreactivity, suggesting a causal relationship between GLYT2 expression and glycine distribution. In this report, we analyzed if such a relationship does exist by using neuronal cultures derived from embryonic spinal cord. GLYT2 was synthesized in a small subpopulation of neurons where it was targeted both to dendrites and to axons, being the axonal content higher than the dendritic one. At early stages in the development of cultured spinal neurons, the highest GLYT2 levels were found in the axonal growth cones. As the culture matured, immunoreactivity extended to the axonal shaft. Double-immunofluorescence experiments indicated a perfect co-localization of GLYT2 and glycine immunoreactivity in cultured neurons. Moreover, the concentration of glycine into neurons expressing GLYT2 was proportional to the concentration of the transporter. This observation was reproduced in GLYT2-transfected COS cells. These evidences indicate that the high content of glycine observed in some neurons in culture is indeed achieved by the concentrative task performed by GLYT2, and that GLYT2 can be used as a reliable marker for identification of glycine-enriched neurons.