In HPLC, temperature programming and isocratic separation were investigated for resolving a mixture of nucleotide polymers. A mixture of short oligonucleotides pd(A)(12)(-)(18) was resolved in less than 4 min by utilizing isocratic separation. The method is sensitive to organic modifier concentration; a 0.5% change of organic modifier in the mobile phase leads to more than doubling of the total analysis time. Temperature programming was used to optimize the separation of larger oligonucleotides pd(A)(25)(-)(30) and pd(A)(40)(-)(60). When the column temperature was changed by programming during the separation, the analysis time was 75% less than for an isocratic/isothermal run. The low amounts of ion-pairing agent in the mobile phase (between 5 and 10 mM) make these methods suitable for electrospray ionization mass spectrometry.