The main mechanism of action of the anticancer drug gemcitabine is assumed to be incorporation of its triphosphate (dFdCTP) into DNA, resulting in inhibition of DNA polymerization, inhibition of DNA synthesis and repair. Another mechanism is inhibition of ribonucleotide reductase leading to imbalance in the deoxyribonucleotide (dNTP) pools. One assay to measure dNTP pools is based on oligonucleotide elongation mediated by DNA polymerase. Since the latter may be affected by dFdCTP, we studied the effect of 0.1-600 pmol dFdCTP on this assay; 10 pmol and more dFdCTP significantly increased the average dpm of the blank (absence of other dNTP) and that of the calibration line of dATP (1.4-1.6-fold); 0.1 pmol and more increased that of the standard dGTP curve significantly (1.1-1.8-fold); 10-75 pmol decreased that of dCTP while 75 and 100 pmol significantly increased that of dCTP (1.3-fold); 50 pmol significantly increased that of dTTP (1.3-1.5-fold). For dATP, dGTP and dTTP, a saturation was reached at 100 pmol dFdCTP, but not yet for dCTP. To minimize these effects, we added an excess of 200 pmol dFdCTP to all samples and calibration lines when measuring dNTP levels of gemcitabine treated samples. In this way the effects of gemcitabine on dNTP levels were studied in human A2780 ovarian, HT29 colon, K562 myelogenous leukemia, H322 non-small cell lung cancer cell lines and the murine lung cancer cell line Lewis Lung. In all cell lines, intrinsic dTTP pools (3-77 pmol/106 cells) were the highest, followed by dATP (1.5-31), dCTP (0.7-27) and (nd-14) dGTP. Exposure to 1 and 10 microM gemcitabine for 4-h concentration dependently decreased dATP 3-10-fold and dGTP to undetectable levels, but dCTP at most 3-fold, while dTTP increased. In conclusion, dFdCTP affects dNTP measurements with the DNA polymerase elongation assay, but its effect could be controlled by addition of similar amounts of dFdCTP to each assay.