A simple and precise method for the simultaneous determination of free D-aspartic acid, D-serine and D-alanine in mouse brain tissues was established, using a reversed-phase HPLC system with widely used pre-column derivatizing reagents, o-phthaldialdehyde and N-t-butyloxycarbonyl-L-cysteine. With the present method, the contents of these three D-amino acids in hippocampus, hypothalamus, pituitary gland, pineal gland and medulla oblongata as well as cerebrum and cerebellum of mutant mice lacking D-amino-acid oxidase activity were determined and compared with those obtained for control mice. In both mice, extremely high contents of D-serine were observed in forebrain (100-400 nmol/g wet tissue), and the contents were small in pituitary and pineal glands. While, D-serine contents in cerebellum and medulla oblongata of mutant mice were about ten times higher than those in control mice. In contrast, D-alanine contents in mutant mice were higher than those in control mice in all brain regions and serum.