An isocratic reversed-phase high-performance liquid chromatographic system is described for the separation of a relatively large number (greater than 20) of ribonucleotides, deoxynucleotides, cyclic nucleotides and deoxycyclic nucleotides. A 25 cm x 5 mm ODS-Hypersil (5 microns particle size) column was used with methanol-triethylammonium phosphate buffer as eluent. The effects of methanol content, pH, and ionic strength of the buffer on retention and resolution of the nucleotides have been studied. The applicability of the system was demonstrated by the analysis of nucleotides in cells and tissue extracts.