Since 3-methoxy-4-hydroxyphenylglycol (MHPG) is a neutral metabolite from norepinephrine, it will be a diagnostic marker for mental diseases such as depression. For the development of an immunoassay, the natural enantiomer of MHPG would be required to prepare its antigen and to examine the specificity of the antibody. A natural enantiomer synthesized, however, has not been obtained so far. In this paper, we attempted to enantioseparate synthetic DL-MHPG and to assign D-enantiomer from the optical rotation of MHPG purified from human urine, because endogenous norepinephrine occurs as D-enantiomer which should metabolically generate D-MHPG. Enantioseparation conditions were tested using a Ceramospher Chiral RU-1 column (4.6 x 250 mm) at a flow rate of 0.5 ml/min. The resolution was adequate for the analysis and purification of synthetic DL- and the urinary MHPGs using methanol as a mobile phase and the column temperature at 0 degrees C, where DL-MHPG was detected as two peaks. The earlier peak (peak 1) showed (-) optical rotation, while the latter gave (+) optical rotation. After being treated with beta-glucuronidase, the normal human urine was extracted with ethyl acetate and then evaporated to dryness. The residue was suspended in water and the supernatant was analyzed and purified by a reversed phase column with a multi channel detector. A peak corresponding to MHPG was collected and concentrated to dryness. The pooled residues were dissolved in methanol and enantioseparated on the chiral HPLC. The urinary MHPG appeared as a single peak which was corresponded to the earlier peak of DL-MHPG and showing (-) optical rotation. Thus, the urinary MHPG was found to be D-(-)-MHPG. Then the absolute configuration of enantioseparated MHPGs were assigned to each optical rotation, judging from the chemical data and the metabolic pathway of the urinary D-MHPG. These enantiomers will be useful for studying on biochemistry and immunoassay.