The cytotoxicity of N3-methyl-5'-deoxy-5-fluorouridine (N3-Me-5'-dFUR), a novel metabolite of the anticancer pro-drug 5'-deoxy-5-fluorouridine (5'-dFUR), has been evaluated by in vitro experiments with cultures of different cancer cell lines. The new metabolic product was found to be non-toxic in all the cell growth experiments performed. The absence of cytotoxicity could be explained by the observation that the metabolite was not recognized as a substrate by thymidine phosphorilase, the enzyme responsible for 5-fluorouracil (5-FU) release from doxifluridine, as ascertained by high-performance liquid chromatography/ultraviolet (HPLC-UV) analysis of the incubation mixture. The biomethylation process leading to N3-Me-5'-dFUR could be considered as a possible detoxification pathway, altering the drug bioavailability, in competition with 5'-dFUR cleavage to the active 5-FU.