A validated and fully automated chiral 2D-HPLC system was developed for the simultaneous determination of N-methyl-d-aspartic acid (NMDA) analogues by combining a long microbore-monolithic ODS column (0.53mm i.d.×1000mm) and narrowbore-enantioselective columns (1.5mm i.d.×150 or 250mm). The target analytes, enantiomers of N-methylaspartic acid (NMA) and N-methylglutamic acid (NMG), were precolumn-derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and detected by their fluorescence. The values of the lower limit of quantification for these enantiomers were 2.5fmol. In the tissues and plasma of rats, neither NMA nor NMG were detected. On the other hand, in the mantle and foot of Scapharca broughtonii, a large amount of NMDA was present (170.1 and 43.5nmol/g), and the enantiomers of NMG were also observed. Meretrix lusoria contained NMDA (29.3nmol/g) and NMLG (13.8nmol/g), and Ruditapes philippinarum contained only NMLG (2.6nmol/g). The obtained results were confirmed using three different enantioselective columns and also using a 2D-HPLC–MS/MS system. These results indicated that neuroactive d-amino acid, NMDA, and its analogues were present in animals, and their physiological significance is expected to be clarified.