Abstract In a clinical study with oral gemcitabine (2′,2′-difluorodeoxycytidine, dFdC), 2′,2′-difluorodeoxyuridine (dFdU) was extensively formed and accumulated after multiple oral dosing. Here, we have investigated the in vitro cytotoxicity, cellular uptake, efflux, biotransformation, and nucleic acid incorporation of dFdC and dFdU. Short-term and long-term cytotoxicity assays were used to assess the cytotoxicity of dFdC and dFdU in human hepatocellular carcinoma HepG2, human lung carcinoma A549, and Madin-Darby canine kidney cell lines transfected with the human concentrative or equilibrative nucleoside transporter 1 (hCNT1 or hENT1), or empty vector. Radiolabeled dFdC and dFdU were used to determine cellular uptake, efflux, biotransformation, and incorporation into DNA and RNA. The compounds dFdC, dFdU, and their phosphorylated metabolites were quantified by high-performance liquid chromatography with UV and radioisotope detection. dFdU monophosphate, diphosphate, and triphosphate (dFdU-TP) were formed from dFdC and dFdU. dFdU-TP was incorporated into DNA and RNA. The area under the intracellular concentration-time curve of dFdC-TP and dFdU-TP and their extent of incorporation into DNA and RNA inversely correlated with the IC50 of dFdC and dFdU, respectively. The cellular uptake and cytotoxicity of dFdU were significantly enhanced by hCNT1. dFdU inhibited cell cycle progression and its cytotoxicity significantly increased with longer duration of exposure. dFdU is taken up into cells with high affinity by hCNT1 and phosphorylated to its dFdU-TP metabolite. dFdU-TP is incorporated into DNA and RNA, which correlated with dFdU cytotoxicity. These data provide strong evidence that dFdU can significantly contribute to the cytotoxicity of dFdC. [Mol Cancer Ther 2008;7(8):2415–25]