The applicability of a new type of anion exchanger, MonoQ HR 5/5, and the Pharmacia-LKB fast protein liquid chromatographic (FPLC) system to the separation of nucleotides is described. The elution characteristics of adenosine-5'-, cytidine-5'-, uridine-5'-, guanosine-5'-mono-, -di- and -triphosphates and inositol-5'-monophosphate reference compounds, and of nucleotides originating from various biological samples, are optimized by varying the concentration gradient programme with ammonium phosphate buffer. Some practical examples of biological interest for monitoring the metabolic changes of nucleotides are presented.