Abstract: A highly sensitive and selective reversed phase high performance liquid chromatography (RP-HPLC) method for the analysis of nucleosides in DNA and RNA of E.coli is described. The analytical column was Supelco Inc.C₁₈ (250 mm×4.5 mm) column(at 20℃). The separation of the nucleosides was achieved in 65 min using a two buffer step gradient system. Buffer A (2.5% v/v methanol, 0.01 mol/L KH₂PO₄, pH 4.6) was pumped for the first 28 min of the run, followed by 37 min of Buffer B (8.0% v/v methanol, 0.01 mol/L KH₂PO₄, pH 4.6). Absorption was measured simultaneously at 254 nm and 280 nm with sensitivity of 0.01 aufs and the flow rate was 1.0 mL/min. The relative standard deviation (RSD) were from 0.86% to 2.62% and the recoveries ranged between 80.20%～99.31%.