A method based on high performance liquid chromatography (HPLC) for the analysis of glucose, ethanol and glycerol simultaneously is presented. The baseline separation was achieved at room temperature (25-30 degrees C) on a Beckman mu-Spherogel carbohydrate column (6.5 mm i.d. x 30 cm) with H2SO4-H2O(0.5:1,000, V/V) as mobile phase at a flow rate of 1.000 mL/min. All the substances were detected with the Beckman 186 Refractive Index Detector. Under these conditions, distinct peaks of glucose, ethanol and glycerol were resolved within 15 min. The retention time in the particular condition was (6.04 +/- 0.04) min for glucose, (13.36 +/- 0.08) min for ethanol and (8.72 +/- 0.05) min for glycerol. The detection limit for glucose, ethanol and glycerol were found to be 10(-5) g, 10(-4) g and 10(-5) g respectively. A series of experiments have been performed to investigate the glucose consumption and the ethanol, glycerol production in the random growth of Saccharomyces cerevisiae with the method above. Only 0.5 mL sample was needed each time. In 30 hours after inoculation, the cell density increased from 10(6) mL-1 to 10(7) mL-1. It was found that the glucose concentration decreased approximately linearly after 8 hours, and ethanol and glycerol was detected after the delay period as expected. The results show that the method proposed is rapid and convenient with enough accuracy. The method may be used to investigate the metabolic behavior of yeast in the fermentation process at the early stage or detect the slight changes of concentrations of some objective substances in particular culture experiments.