Two hydroxylated metabolies (M1 and M2) have been isolated from rabbit urine after administration of Ftorafur (FT). The structures of 3'-OH-FT and 4'-OH-FT were assigned to M1 and M2, respectively. A reverse-phase high performance liquid chromatography assay was developed for jeasuring FT, M1, M2, and 5-fluorouracil (FU) plasma levels. M1, M2, and FU were present in rabbit and rat plasma in greatly varying concentrations after FT administration. Pharmacokinetic studies suggest that FU formation proceeds via metabolic intermediate(s) and that the extent of FT activation is variable. A horse liver thymidine phosphorylase ,reparation capable of catalyzing the conversion of beta-ribo-2'-deoxy-5-fluorouracil to FU was inactive against FT and M1. However, 20% of M2 was converted to FU by this enzume, which suggests that the urinary metabolite M2 consisted of a mixture of enantiomers with 20% present in the natural beta-D configuration. The stereochemistry of M1 remains unknown. Hydroxylation of FT to beta-D-4'-OH-FT and subsequent cleavage to FU by thymidine phosphorylase represents one possible activation mechanism of FT to FU. ,owever, lack of correlation between plasma levels of M2 and FU indicates that this mode of metabolic activation may account for only part of the overall activation of FT in vivo.