Exponentially growing K562 cells incubated with 1-beta-D-arabinofuranosylcytosine (ara-C) accumulate ara-C triphosphate (ara-CTP) at a higher rate and to a greater concentration after pretreatment with 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) than do cells treated with ara-C alone. Potentiation of ara-C metabolism is due in part to an indirect effect of F-ara-A triphosphate (F-ara-ATP)-mediated reduction in deoxynucleotide pools and consequent activation of deoxycytidine kinase. Because the levels of deoxynucleotide pools and the activity of deoxycytidine kinase are cell cycle-specific, we investigated the effect of cell cycle phases on the accumulation of ara-CTP and the influence of F-ara-A pretreatment on such accumulation. Exponentially growing K562 cells were fractionated into G1, S, and G2+M phase-enriched subpopulations (each enriched by > 60%) by centrifugal elutriation. The rate of ara-CTP accumulation was 22, 25, and 14 microM/h and the rate of F-ara-ATP accumulation was 38, 47, and 33 microM/h in the G1, S, and G2+M subpopulations, respectively. The rate of elimination of arabinosyl triphosphates was similar among the different phases of the cell cycle. After pretreatment with F-ara-A, the rate of ara-CTP accumulation in the G1, S, and G2+M phase-enriched subpopulations was 43, 37, and 26 microM/h, indicating a 1.7-, 1.5-, and 1.9-fold increase, respectively. These results suggest that a combination of F-ara-A and ara-C may effectively potentiate ara-CTP accumulation in all phases of the cell cycle. This observation is consistent with the results of studies on the modulation of ara-C metabolism by F-ara-A in lymphocytes and leukemia blasts obtained from patients with chronic lymphocytic leukemia and acute myelogenous leukemia, respectively.