D-Serine, an endogenous and obligatory coagonist for the glycine site of the N-methyl-D-aspartate receptor in mammals, is synthesized from L-serine by serine racemase. Serine racemase and D-serine have long been believed to occur predominantly in astrocytes, according to immunohistochemical studies. Recent studies have demonstrated, however, that both the mRNA and protein levels of serine racemase are considerably higher in neurons than in astrocytes in primary cultures of the rat brain and that the mRNA level of serine racemase predominates in neurons of the adult rat brain. Here we report the application of in situ hybridization based on tyramide signal amplification for the detection of serine racemase mRNA in sections of the adult rat retina and optic nerve head. The localization of serine racemase mRNA could be demonstrated in ganglion cells, amacrine cells, bipolar cells, horizontal cells, and Müller cells of the retina as well as in the astrocytes of the optic nerve head and the lamina cribrosa. This is the first study to demonstrate the exact localization of serine racemase mRNA at the cellular or tissue level in the retina and the optic nerve head. These results suggest that both the neuron- and glia-derived D-serine could modulate neurotransmission via the glycine site of the N-methyl-D-aspartate receptors in the retina.