The concurrent determination of free amino acid enantiomers and non-chiral amino acids in rat brain and serum was accomplished by high-performance liquid chromatography with fluorimetric detection after derivatization with N-tert.-butyloxycarbonyl-L-cysteine and o-phthaldialdehyde. The method revealed the presence of a large amount of free D-serine (0.22 mumol/g of tissue; D/D + L ratio = 0.25) in the brain whereas D-aspartate and D-alanine were established to be at trace levels. These results further support the presence of D-serine in adult brain tissues as demonstrated by recent work using gas chromatography.