Gemcitabine (2', 2'-difluorodeoxycytidine, dFdC) is a deoxycytidine (dCyd) analog that extensively modulates intracellular CTP and dCTP metabolism. In Chinese hamster ovary (CHO) cells, a 4-hour exposure to gemcitabine (100 mumol/L) reduced cellular CTP and dCTP concentrations to 5.9% and 50%, respectively. Intracellular UTP concentrations increased, indicating a metabolic block at CTP synthetase. Pool-sizes of ATP and GTP remained unaffected. In contrast, a CHO mutant deficient in deoxycytidine kinase, and thus unable to accumulate dFdCTP, maintained its CTP pools under identical conditions, suggesting that the CTP pool depletion was dependent on dFdC phosphorylation. Neither 100 mumol/L arabinosylcytosine nor 5 mmol/L hydroxyurea affected CTP levels, indicating that inhibition of DNA synthesis by analog incorporation or by depletion of dNTP pools were not the causes of the CTP pool perturbation. Metabolic studies demonstrated that incorporation of [3H] uridine into the UTP pool was not impaired by dFdC treatment, whereas the specific activity of the CTP pools decreased as a function of increasing gemcitabine concentration and time of exposure. Comparable results were obtained using 3-deazauridine, a known inhibitor of CTP synthetase. We conclude that high cellular concentrations of dFdCTP deplete cellular CTP concentrations by inhibition of the dCTP pool and also may be a limiting factor for RNA synthesis.