The present paper describes a quantitative determination method of tetracyclines by reversed phase high performance liquid chromatography. The method used a Waters 10 C18 4.6 nm ID x 250 nm reversed phase column, with a mobile phase composed of 14% acetonitrile-3% dimethylformamide-83% water containing 0.02 mol/L citric acid (pH-2.5), the detector wavelength was set at 350 nm, and the flow rate was maintained at 1.0 ml/min. Demethylchlortetracycline, epi-demethylchlortetracycline, demethyltetracycline, epi-demethyltetracycline, tetracycline, epi-tetracycline, chlortetracycline and epi-chlortetracycline can be separated highly efficiently. The tetracyclines and their anhydro- products can be separated on the same column with 15% acetonitrile- 15% dimethylformamide- 70% 0.02 mol/L citric acid as the mobile phase, and detected at UV 270 nm.