The method for the simultaneous determination of thiamine hydrochloride, pyridoxine hydrochloride and cyanocobalamin by high-performance liquid chromatography (HPLC) with coulometric electrochemical and UV detections is presented. The retention time of vitamins was repeatedly determined by isocratic elution using 0.05 M phosphate buffer-10% methanol and 0.018 M trimethylamine (1 ml min(-1), pH 3.55) as mobile phase with the Supelco LC 18 column 5 microm (25 cm x 4.6 mm). The specificity of the method was demonstrated by the retention characteristics, coulometric electrochemical and UV detection. The limits of detection of thiamine, pyridoxine and cyanocobalamin were: 9.2, 2.7 and 0.08 ng/ml, respectively. The method was characterized also by wide concentration range, high sensitivity and good accuracy (99.6-102.7%). The repeatability of the method was evaluated at different level of concentration of vitamins and the relative standard deviation was below 4.5%. The method was successfully applied for the quantification of Vitamins B1, B6 and B12 in pharmaceutical preparations and dietary supplements.