In the developing central nervous system (CNS), the activation of N-methyl-D-aspartate (NMDA) receptors is probably an important regulator of processes such as synaptogenesis and neurite growth. NMDA receptor activation is dependent upon the homeostasis of glutamate and the presence of co-agonists such as D-serine. We have investigated the expression of the glutamate transporter excitatory amino acid transporter-1 (EAAT1 or GLAST) as the key regulator of retinal extracellular glutamate levels, and the ontogeny of D-serine expression in the developing human retina. The expression of EAAT1 and D-serine was compared to the temporal and spatial distribution of the synaptic vesicle marker synaptophysin and the synaptic vesicle glutamate transporter vGLUT1. We also examined the co-expression of EAAT1 and cellular retinaldehyde-binding protein (CRALBP), and the co-expression of EAAT1 and D-serine. Human retinae aged 10-20 weeks' gestation (WG) were prepared for immunocytochemistry or for Western blotting. Expression of EAAT1 was evident at 10 WG in cell bodies, processes and end-feet of radial glia-like cells at all retinal eccentricities. D-serine immunolabelling was also evident in radial glia-like cells by 12 WG. In contrast, immunoreactivity for synaptophysin only started to appear in the central retina at 12 WG whilst immunoreactivity for vGLUT was slightly later. EAAT1 and d-serine were co-localised to the same cell population. In addition, EAAT1 and CRALBP were also co-localised to the same cell population of radial glia-like cells, suggesting that the EAAT1 and D-serine-positive cells may be Müller cells. This study shows that key potential modifiers of NMDA receptor activity are present before synaptic vesicle proteins are evident and may thus play a role in shaping synaptogenesis in the developing human retina.