The accumulation of cytosine arabinoside-5'-triphosphate (ara-CTP), the activities of nucleotide-metabolizing enzymes and the nucleoside transport capacity were examined in eleven human leukemic cell lines differing in phenotype. The highest amount of ara-CTP was accumulated in T-acute lymphoblastic leukemia cells (T-ALL), followed by myeloid leukemia cell lines (AML), and the least accumulation was observed in common acute lymphoblastic leukemia (c-ALL) and B-acute lymphoblastic leukemia cells (B-ALL). The levels of enzymes involved in ara-C metabolism (deoxycytidine kinase, pyrimidine monophosphate kinase and deoxycytidylate deaminase) did not correlate with ara-CTP accumulation. The sensitivity of the leukemic cell lines to ara-C, which was measured in terms of decrease of clonogenic survival, correlated with the amount of ara-CTP formed. Moreover, the nucleoside transport capacity, estimated from the binding of the radiolabeled nucleoside analogue, [3H]nitrobenzylthioinosine, showed a good correlation with ara-CTP accumulation. The mean numbers of nucleoside-binding sites in T-ALL cells were significantly greater than in B-ALL cells. We conclude that the cellular nucleoside transport capacity is the most important factor for the formation and accumulation of ara-CTP in the cells.