d-serine is the primary NMDA receptor (NMDAR) co-agonist at mature forebrain synapses and is synthesized by the enzyme serine racemase (SR). However, our understanding of the mechanisms regulating the availability of synaptic d-serine remains limited. Though early studies suggested d-serine is synthesized and released from astrocytes, more recent studies have demonstrated a predominantly neuronal localization of SR. More specifically, recent work intriguingly suggests that SR may be found at the postsynaptic density, yet the functional implications of postsynaptic SR on synaptic transmission are not yet known. Here, we show an age-dependent dendritic and postsynaptic localization of SR and d-serine by immunohistochemistry and electron microscopy in mouse CA1 pyramidal neurons. In addition, using a single-neuron genetic approach in SR conditional knockout mice from both sexes, we demonstrate a cell-autonomous role for SR in regulating synaptic NMDAR function at Schaffer collateral (CA3)-CA1 synapses. Import...