Intracellular accumulation of triphosphorylated derivatives is essential for the cytotoxic activity of nucleoside analogues. Different mechanisms opposing this accumulation have been described. We have investigated the dephosphorylation of monophosphorylated fludarabine (F-ara-AMP) by the purified cytoplasmic 5'-nucleotidase cN-II using HPLC and NMR. These studies clearly showed that cN-II was able to convert F-ara-AMP into its non phosphorylated form, F-ara-A, with a Km in the millimolar range and Vmax = 35 nmol/min/mg, with both methods. Cytoplasmic 5'-nucleotidase cN-II can degrade this clinically useful cytotoxic nucleoside analogue and its overexpression is thus likely to be involved in resistance to this compound.