A reversed-phase gradient elution system with methanol-triethylammonium phosphate buffer (83.3 mM, pH 6.0) as eluent on C(18)-bonded silica is described for the separation of 38 ribonucleotides, deoxynucleotides, cyclic nucleotides and deoxycyclic nucleotides in less than 33 min. The retention of the nucleotides can be precisely controlled by adjusting the pH, buffer concentration and methanol content in the mobile phase. The system is especially useful for the analysis of low levels of cyclic nucleotides in cells and tissues.