D-Serine, a co-agonist at the NMDA receptor (NMDAR), is synthesized from L-serine by the enzyme serine racemase (SR), which is heavily expressed in the forebrain. Although SR was originally reported to be localized exclusively to astrocytes, recent conditional knock out results demonstrate that little SR is expressed in forebrain astrocytes. As a consequence, the cellular location of its product, D-serine, in the brain is also uncertain. Immunocytochemistry now indicates that SR is expressed primarily in forebrain glutamatergic neurons with the remainder in GABAergic interneurons. We utilized SR deficient (SR-/-) mice, which have <15 % of normal D-serine levels, to validate and optimize a D-serine immunohistochemical method. Nearly all of the D-serine in neocortex and hippocampus (HP) is found in neurons, with virtually no D-serine co-localizing with two astrocyte markers. Interestingly, only a subset of the D-serine positive neurons contained SR in the neocortex and HP. Greater than half of the D-serine positive neurons were GABAergic interneurons, with a majority of these neurons containing parvalbumin and/or somatostatin. Only ~25-40 % of interneurons expressed SR in the neocortex and HP. Finally, we demonstrate in human post-mortem neocortex that SR is found in both excitatory and inhibitory neurons, but not in S100β-containing astrocytes. In sum, these findings conclusively demonstrate that the majority of D-serine is both synthesized and stored in neurons. It will be important to determine the functional significance for the separation of synthesis and storage of D-serine in neurons, as well as the presence of this NMDAR co-agonist in GABAergic interneurons.