Because of the use of HPLC for the determination of free nucleotides, nucleosides, and bases in samples of biological origin, investigations that were very difficult or impossible to perform only a decade ago are now possible. Microparticulate, totally porous, chhemically bonded particles appear to be the column packings that will be used routinely in the future because analyses are achieved with high sensitivity, selectivity, efficiency, and speed. These packings are stable, the results are reproducible, and the data are quantitative. In addition, the reversed-phase mode of HPLC offers improvements over the ion-exchange mode for the analysis of nucleosides, bases, and other UV-absorbing compounds which are not ionic. However, the ion-exchange mode may be used as a complementary method for very polar or ionic compounds which are eluted too rapidly with reversed phase. A sample can be chromatographed with each mode and a profile--a complete picture of nucleotide, nucleoside, and base levels in the sample--obtained. Alternatively, ion pairing can be used with the reversed-phase mode; thus both the ionic and lipophilic compounds can be determined in one separation.