A determination method for individual natural vitamin B(6) compounds was developed. The vitamin B(6) compounds were specifically converted into 4-pyridoxolactone (PAL), a highly fluorescent compound, through a combination of enzymatic reactions and HCl-hydrolysis. PAL was then determined by HPLC. Pyridoxal was completely oxidized to PAL with pyridoxal 4-dehydrogenase (PLDH). Pyridoxine and pyridoxamine were totally converted into PAL through a coupling reaction involving pyridoxine 4-oxidase and PLDH, and one involving pyridoxamine-pyruvate aminotransferase and PLDH, respectively. The 5'-phosphate forms and pyridoxine-beta-glucoside were hydrolyzed with HCl, and then determined as their free forms. Pyridoxine 5'-phosphate and pyridoxine-beta-glucoside were not separately determined here. Three food samples were analyzed by this method.