A novel method for the determination of vitamin B12 by high-performance liquid chromatography with fluorescence detection is reported. The method was simple and highly sensitive with good precision. Vitamin B12 was analyzed by HPLC on a muBondapak C18 column (300x3.9 mm, 10 microm) with methanol-water (30:70) as mobile phase and fluorescence detection at 305 nm (with excitation at 275 nm). The calibration graph was linear from 1.000 to 100.0 ng ml(-1) for vitamin B12 with a correlation coefficient of 0.998 (n=6). The detection limit was 0.1 ng ml(-1). The method was successfully applied to the determination of vitamin B12 in vitamin B12 tablets, multivitamin tablets and fermentation medium. The recovery was from 94 to 102% and the relative standard deviation was in the range of 1.8 to 4.1%.