Abstract The retention behaviour of nucleobases (purine and pyrimidine bases) and their nucleosides in systems consisting of water-ethanol mixtures with added electrolytes as mobile phase and a resin-type anion exchanger (Aminex A-28) as stationary phase has been systematically investigated. The effect of pH, type and concentration of the counterion, ethanol content of the mobile phase and temperature on the retention and column efficiency has been determined. The results obtained show that all of these parameters have a significant effect and have to be optimized in order to separate the nucleobases and nucleosides by isocratic elution. The separation of fourteen compounds has been achieved in ca. 30 min. The ability of the phase system is demonstrated by the analysis of a calf thymus DNA hydrolyzate.