Evidence of in vivo biomethylation of the anticancer pro-drug 5′-deoxy-5-fluorouridine (5′-dFUR) is presented for the first time. Biomethylation seems to occur specifically at the N 3 site on the pyrimidine ring. This novel metabolic product was detected by gas chromatography-mass spectrometry of the trimethylsilylated extract of plasma samples from cancer patients undergoing doxifluridine chemotherapy. Considering the observed electron impact fragmentation pattern, the metabolic product was tentatively identified as {ce:inline-formula}N 3-Me-5′-dFUR{/ce:inline-formula}. Definite confirmation of the proposed structure was achieved by comparison of the mass spectra and chromatographic characteristics of the suspected metabolite with those of a synthetically prepared reference standard.