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Purine nucleoside antimetabolites, such as clofarabine, are effective antileukemic agents. However, their effectiveness depends on an initial activation step in which they are monophosphorylated by deoxycytidine kinase (dCK). Some purine nucleoside antimetabolites and their monophosphate derivatives are exported by the ABC transporter ABCG2. Because clofarabine is a dCK substrate, and we show substantial variation in dCK and ABCG2 in myeloid leukemia, we hypothesized that the activity of dCK may modulate ABCG2-mediated resistance to clofarabine by regulating the formation of clofarabine monophosphate. We show that ABCG2 influence on clofarabine cytotoxicity was markedly influenced by dCK activity. When dCK expression was reduced by siRNA, clofarabine cytotoxicity was strongly reduced by enhanced ABCG2-mediated efflux. Conversely, dCK overexpression blunted ABCG2-mediated efflux of clofarabine by increasing the formation of clofarabine nucleotides. The use of an ABCG2 inhibitor confirmed that ABCG2 export of clofarabine is maximal when dCK levels are minimal. Analysis of intracellular clofarabine metabolites suggested that ABCG2 exported clofarabine more readily than clofarabine monophosphate. That ABCG2 primarily effluxes clofarabine, but not chlorfarabine-monophosphate, was confirmed by HPLC analysis of drug exported from ABCG2-overexpressing cells. Because the level and function of dCK and ABCG2 vary substantially among other types of cancer, these findings have important implications not only for clofarabine therapy but for purine nucleoside therapy in general. Therefore, we propose that addition of ABCG2 inhibitors would effectively increase the antitumor efficacy of purine nucleosides by blocking drug efflux that may be a significant mode of resistance when dCK levels are low. Cancer Res; 71(5); 1781–91. ©2011 AACR.