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Gemcitabine is a pyrimidine analog effective against many solid tumors. Following intravenous administration, deaminases in the plasma rapidly convert the parent compound, gemcitabine, to its deaminated metabolite, 2′,2′-difluorodeoxyuridine (dFdU), resulting in an elimination half-life for gemcitabine of 8 min. The half-life of dFdU, however, is upwards of 14 h, yielding plasma concentrations that are frequently 10–20-fold higher than that of gemcitabine. The uptake of gemcitabine into tumor cells is facilitated by both concentrative (hCNT) and equilibrative (hENT) nucleoside transporters. Recently, it was observed that dFdU is a substrate for hCNT as well. The purpose of this study was to investigate the effects of dFdU on gemcitabine uptake and efflux via hENT1 and hENT2 in HeLa cells. Our results suggest that dFdU is a substrate for both hENT1 and hENT2 as well as a competitive inhibitor of gemcitabine transport at concentrations >100-fold lower than those typically achieved in plasma (IC50 = 0.45 and 1.2 μM for hENT1/2 and hENT2, respectively). However, inhibition of gemcitabine uptake is time-dependent, as dFdU limits gemcitabine uptake into HeLa cells by more than 80% during short (<20 s) incubation periods but increases net gemcitabine retention as incubation length increases. While dFdU enhances the accumulation of gemcitabine by up to 1.5-fold following a 60 min incubation, dFdU did not enhance gemcitabine cytotoxicity. In conclusion, this is the first report of an interaction between dFdU and gemcitabine suggesting that the deaminated metabolite may play an important role in the disposition of gemcitabine in tumor cells.