We attempt to identify the plasma membrane transporter involved in the uptake of 5Ј-deoxy-5-fluorouridine (5Ј-DFUR), an intermediate metabolite of capecitabine. This novel oral fluoropyrimidine is used in cancer treatments and is a direct precursor of the cytostatic agent 5Ј-fluorouracil. We also examine the role of the transporter in 5Ј-DFUR cytotoxicity. The human concentrative nucleoside transporter (hCNT1) was cloned from human fetal liver and expressed in Xenopus laevis oocytes. The two-electrode voltage-clamp technique was used to demonstrate that 5Ј-DFUR, but not capecitabine or 5Ј-FU, is an hCNT1 substrate. Then, hCNT1 was heterologously expressed in the mammalian cell line Chinese hamster ovary-K1. Functional expression was demonstrated by monitoring transport of radiolabeled substrates and by using a monospecific polyclonal antibody generated against the transporter. hCNT1-expressing cells were more sensitive to 5Ј-DFUR than vector-transfected or wild-type cells.