Recently, a substantial amount of free D-serine has been demonstrated in rat brain, although it has long been presumed that D-amino acids are uncommon in mammals. The anatomical distribution and age-related changes in endogenous D-serine have been examined here to obtain insight into its physiological functions. Free D-serine exclusively occurs in brains, with a persistent high content from birth to at least 86 postnatal weeks. The patterns of the regional variations and the postnatal changes in brain D-serine are closely correlated with those of the N-methyl-D-aspartate (NMDA)-type excitatory amino acid receptor. Because D-serine potentiates NMDA receptor-mediated transmission by selective stimulation of the strychnine-insensitive glycine site of the NMDA receptor, it is proposed that D-serine is a novel candidate as an intrinsic ligand for the glycine site in mammalian brain.