The use of Reversed-Phase High Performance Liquid Chromatography(RP-HPLC) was investigated for the separation of oligonucleotides. By the gradient elution with the mobile phase of methanol, 0.1M KH₂PO₄ and 0.02 M MaCl₂, the mixtures could be separated in a 250 mm × 4.6 mm I.D. column. The number of theoretical plates and resolution were compared between this technique and Micellar Electrokinetic Capillary Chromatography(MECC). The charged nucleic acid constituents in the RP-HPLC were relatively better separated than in the MECC.