D-Serine, the endogenous ligand for the glycine modulatory binding site of the NMDA receptor, and serine racemase, the enzyme that converts L-serine to D-serine, have been reported in vertebrate retina; initial reports suggested that localization was restricted to Müller glial cells. Recent reports, in which D-serine and serine racemase were detected in neurons of the brain, prompted the present investigation of neuronal expression of D-serine and serine racemase in retina and whether expression patterns were developmentally regulated. RT-PCR, in situ hybridization, western blotting, immunohistochemistry, and immunocytochemical methods were used to localize D-serine and serine racemase in intact retina obtained from 1 to 3 day, 3 week, and 18 week mouse retinas and in primary ganglion cells harvested by immunopanning from neonatal mouse retina. Results of these analyses revealed robust expression of D-serine and serine racemase in ganglion cells, both in intact retina and in cultured cells. The levels appear to be developmentally regulated with D-serine levels being quite high in ganglion cells of neonatal retinas and decreasing rapidly postnatally. Serine racemase levels are also developmentally regulated, with high levels detected during the early postnatal period, but diminishing considerably in the mature retina. This represents the first report of neuronal expression of D-serine and serine racemase in the vertebrate retina and suggests an important contribution of neuronal D-serine during retinal development.