High levels of d-serine occur in mammalian brain, where it appears to be an endogenous ligand of the glycine site of N-methyl-d-aspartate receptors. In glial cultures of rat cerebral cortex, d-serine is enriched in type II astrocytes and is released upon stimulation with agonists of non-N-methyl-d-aspartate glutamate receptors. The high levels of d-serine in discrete areas of rat brain imply the existence of a biosynthetic pathway. We have purified from rat brain a soluble enzyme that catalyzes the direct racemization of l-serine to d-serine. Purified serine racemase has a molecular mass of 37 kDa and requires pyridoxal 5′-phosphate for its activity. The enzyme is highly selective toward l-serine, failing to racemize any other amino acid tested. Properties such as pH optimum, K_m values, and the requirement for pyridoxal phosphate resemble those of bacterial racemases, suggesting that the biosynthetic pathway for d-amino acids is conserved from bacteria to mammalian brain.