DNA base composition was determined by reversed-phase high-performance liquid chromatography (HPLC). DNA was hydrolysed into nucleosides with nuclease P1 and bacterial alkaline phosphatase. The mixture of nucleosides was applied to HPLC without any further purification. One determination by chromatography needed 2 µg of hydrolysed nucleosides and took only 8 min. The relative standard error of nucleoside analysis was less than 1%. The system described here gives a direct and precise method for determining DNA base composition.