The origin of D-serine was investigated using microdialysis probes to administer radiolabeled glucose, glycine, and L-serine directly into rat brain. In these experiments the labeling of D-serine was found to be determined only by the radioactivity present in the L-serine pool, regardless of the precursor employed, indicating that L-serine is the direct precursor of the D-isomer. Its rate of synthesis was 4.6 +/- 1.2 %/h; 9.2 nmol/g/h). This rate of synthesis is in agreement with that found in the mouse after a loading dose of intraperitoneally injected L-[3H]-serine (4.1 %/h). These rates are also consistent with the degradation rates of D-serine in rat and mouse brain, determined in pulse labeling experiments (4.1 and 3.8 %/h, respectively). Synthesis within the brain from L-serine therefore is adequate to account for the turnover of the brain D-serine pool: contributions from other sources, including the diet, must be minimal. Independence from dietary sources was also demonstrated by the failure of labeled D-serine administered in the drinking water to label the brain pool unless very high doses were given. These results suggest that D-serine in the brain is formed directly by the racemization of L-serine.