D-Amino acids, the enantiomers of L-amino acids extensively present in life systems, have been found in mammals and they are increasingly recognized as physiologically active substances. Especially, D-Ser is related to the learning and memory in the hippocampus and cerebellum, and D-Asp is involved in regulating the hormonal secretion and is thought to be beneficial for healthy skin. According to these reports, determination of D-amino acids in the foodstuffs is expected to develop the functional and health foods and beverages containing D-amino acids. Therefore, in the present study, D-amino acids in the Japanese traditional amber rice vinegar fermented in the earthenware jar, Kurozu, are determined and the alterations of D-amino acid amounts during the fermentation processes are investigated. Kurozu samples were diluted by adding H2O and a portion of the aliquot was used for the derivatization of amino acids with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). NBD-derivatives were determined by using the 2D-HPLC-FL/MS/MS system combining a microbore-monolithic ODS-column (0.53 mm i.d. x 1000 mm) and the narrowbore-Pirkle-type enantioselective column (1.5 mm i.d. x 250 mm). By using this system, large amount of D-Ala, relatively large amounts of D-Asp, D-Glu, D-Leu, D-Ser, and small amount of D-allo-Ile were found in the commercially available Kurozu product (D-Ala, 3848; D-Asp, 252; D-Glu, 412; D-Leu, 42; D-Ser, 80; D-allo-Ile, 17 nmol/mL). Concerning the changes of the D-amino acid amounts during fermentation, the levels of D-Ala, D-Asp, D-Glu and D-Ser gradually increased during 4 months. The amount of D-Leu increased rapidly within 1 month. These results indicate that Kurozu is a useful material for the functional beverage containing abundant D-amino acids, and the investigations about the origin and function of D-amino acids in Kurozu are in progress.