A rapid and reliable protocol for the simultaneous separation of ribo-, deoxyribo- and cyclic nucleotides has been developed using high-performance liquid chromatography on a C18 microBondapak column and isocratic elution with ammonium phosphate buffer 0.2 m, pH 5.1). Resolution of deoxyribonucleotides has been confirmed by performing resolution before and after periodate oxidation. The general order of elution is ribonucleotides, deoxyribonucleotides and cyclic nucleotides. While periodate oxidation improved the clarity of separation of deoxyribonucleotides by eliminating ribonucleotides, incorporation of methanol in the eluent shortened the retention time of cyclic nucleotides. The application of this method to a complex biological system is reported.