To obtain insight in mechanisms of action of extracellular adenosine triphosphate (ATP) and adenosine, a simple HPLC method has been optimized and applied to investigate ATP metabolism in human whole blood ex vivo. This method provided good chromatographic resolution and peak shape for all eight compounds within a 19 min run time. The baseline was clean, the lower limit of quantification was below 0.3 micromol/L for all adenine nucleotides and the method demonstrated good linearity. Within-day precision ranged from 0.7 to 5.9% and between-days from 2.6 to 15.3%. Simplicity and simultaneous detection of ATP and its metabolites make this method suitable for clinical pharmacokinetic studies.