Pyrimidine nucleotide pools were investigated as determinants of the rate of phosphorylation of 1-beta-D-arabinofuranosylcytosine (ara-C) by Ehrlich ascites cells and cell extracts. Cells were preincubated for 2 h with 10 microM pyrazofurin, 10 mM glucosamine, 50 microM 3-deazauridine, or 1 mM uridine in order to alter the concentrations of pyrimidine nucleotides. Samples of the cell suspensions were taken for assay of adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate (UTP), cytidine 5'-triphosphate, guanosine 5'-triphosphate, deoxycytidine 5'-triphosphate (dCTP), and deoxythymidine 5'-triphosphate; then 1 microM [3H]ara-C was added and its rate of intracellular uptake was measured for 30 min. 3-Deazauridine lowered dCTP and stimulated ara-C uptake; however, pyrazofurin and glucosamine were potent inhibitors of ara-C uptake although they also decreased dCTP levels. Uridine stimulated ara-C uptake despite an increase in dCTP. A crude cytoplasmic extract was prepared by a procedure which permitted results of ara-C kinase assays to be expressed as pmol per min per 10(6) cells as in the cellular uptake studies. When assayed in the presence of mixtures of ribo- and deoxyribonucleoside triphosphates at concentrations close to their cellular levels, ara-C kinase activity closely approximated the cellular uptake rate for the five incubation conditions. Deletion of cytidine 5'-, guanosine 5'-, or deoxythymidine 5'-triphosphate from the assay mixture had little effect, while deletion of dCTP increased kinase activity 9-fold. Elimination of ATP also did not alter kinase activity in the presence of the remaining five ribo- and deoxyribonucleoside triphosphates; however, deletion of UTP reduced activity to 22% of the rate with the control mixture. When ara-C kinase was assayed with only 3 mM ATP, dCTP was a very potent inhibitor (50% inhibition concentration = 0.4 microM). Inhibition was complete at 10 microM dCTP, a concentration below the intracellular dCTP level in control cells (25 microM). With 0.9 mM UTP, enzyme activity was 2-fold greater in the absence of dCTP and the dCTP was 15-fold less potent as an inhibitor (50% inhibition concentration = 6 microM). We conclude that the actual phosphate donor for the phosphorylation of 1 microM ara-C in Ehrlich cells is UTP and not ATP. These observations suggest that successful combination protocols aimed at stimulating ara-C uptake by means of a decrease in dCTP levels must simultaneously preserve or increase UTP pools.