A fluorometric method for the estimation of the vitamin B1 content in food and biological materials is described. Acid hydrolysis is followed by enzymatic treatment to liberate the vitamin B1 of the sample. Interfering substances in the extract are removed to a great extent by chromatography on Amberlite CG 50, a weakly acidic cation exchanger. After oxidation and extraction into an organic medium, the fluorescence of thiochrome is measured. The procedure is simple, reproducible and not susceptible to interference. The recoveries are good.