-L-Dioxolane-cytidine (L-OddC, Troxacitabine, BCH-4556), a novel L-configuration deoxycytidine analog, is under clinical trials for treating cancer. The cytotoxicity of L-OddC is dependent on the amount of the triphosphate form (L-OddCTP) in nuclear DNA. Phosphoglycerate kinase (PGK), a downstream protein of hypoxia-inducible-factor-1␣ (HIF-1␣), is responsible for the phosphorylation of the diphosphate to the triphosphate of L-OddC. In this study, we studied the impact of hypoxia on the metabolism and the cytotoxicity of L-OddC and -D-2Ј,2Ј-difluorodeoxycytidine (dFdC) in several human tumor cell lines including HepG2, Hep3B, A673, Panc-1, and RKO. Hypoxic treatment induced the protein expression of PGK 3-fold but had no effect on the protein expression of APE-1, dCK, CMPK, and nM23 H1. Hypoxic treatment increased L-OddCTP formation and incorporation of L-OddC into DNA, but it decreased the uptake and incorporation of dFdC, which correlated with the reduction of hENT1, hENT2, and hCNT2 expression. Using a clonogenic assay, hypoxic treatment of cells made them 2-to 3-fold more susceptible to L-OddC but not to dFdC after exposure to drugs for one generation. Dimethyloxallyl glycine enhanced the cytotoxicity of L-OddC but not dFdC in Panc-1 cells under normoxic conditions. Overexpression or down-regulation of PGK using transient transfection of pcDNA5-PGK or inducible shRNA in RKO cells affected the cytotoxicity of LOddC but not that of dFdC. The knockdown of HIF-1␣ in inducible shRNA in RKO cells reduced the cytotoxicity of LOddC but not dFdC under hypoxic conditions. In conclusion, hypoxia is an important factor that may potentiate the activity ofis a novel L-configuration deoxycytidine analog with anticancer and antiviral (hepatitis B virus and human immunodeficiency virus) activity (Kim et al., 1992;Grove et al., 1995;Grove and Cheng, 1996;Gourdeau et al., 2004). It was shown in clinical evaluation to be effective against both leukemias and solid tumors. Phase II clinical studies have shown that L-OddC has significant antileukemic activity in patients with acute myeloid leukemia or patients with chronic myelogenous leukemia in the blastic phase (Giles et al., 2002) and modest activity in advanced pancreatic adenocarcinoma (Lapointe et al., 2005). It is currently undergoing phase I/II clinical trials for the treatment of solid tumors.In previous studies, we demonstrated that L-OddC can be phosphorylated by deoxycytidine kinase (dCK) to its monophosphate metabolite, which is further phosphorylated to the di-and triphosphate metabolites by cellular kinases (Grove and Cheng, 1996). The triphosphate metabolite of L-OddC can be incorporated into DNA in vitro by DNA polymerases ␣, , ␦, ␥, and (Kukhanova et al., 1995). Because L-OddC lacks a hydroxyl group at the 3Ј-position, once incorporated into DNA, it causes premature termination of DNA replication and eventually leads to cell death. Therefore, the cytotoxicity of L-OddC is dependent on the steady-state level of the incorporated L-OddC in nuclear DNA. ...