An anion-exchange high performance liquid chromatography (HPLC) method is described for the quantitation of intracellular purine and pyrimidine nucleotides. With an ammonium phosphate salt and pH gradient, complete separation is achieved of all major nucleotides and several interfering substances, such as dehydroascorbic acid and NAD. For optimal resolution of the monophosphates, strict control of the equilibration pH is essential. To prevent interference by a degradation product of NADPH with the determination of GDP, the pH of the high-ionic strength buffer has to be in the range of 4.9-5.0. The use of radially compressed, prepacked cartridges filled with Partisil-10 SAX appeared to be a fast and cheap alternative for expensive stainless-steel columns. The use of ammonium phosphate buffers, in combination with precolumns filled with pellicular silica and SAX resin, and interim EDTA washes prevents baseline shift. This allows analysis at 0.01 Absorbance Units Full Scale during the entire column lifetime (about 180 analyses), which is sufficiently sensitive for the quantitation of low levels of nucleotides, especially when the amount of sample is limited. The usefulness of the presented chromatographic system is demonstrated by the quantitation of the nucleotides in extracts of lymphocytes and neutrophils from the blood of healthy human donors. With this method nucleotide concentrations were measured, with a within-assay variation of 5-10% and an inter-donor variation of 10%.