Abstract Nuclei were prepared from Chinese hamster ovary cells by fractionation in nonaqueous media. The distribution of the four common deoxyribonucleoside triphosphates between the nucleus and the cytoplasm was determined in cells during G1 and S phases and after inhibition of DNA synthesis by hydroxyurea or thymidine. During the G1 phase all triphosphates were present in both the cytoplasm and the nucleus. The distribution between the two compartments differed considerably for the four deoxyribonucleotides. The largest amounts of dATP, dCTP, and dGTP were found in the cytoplasm, whereas most of the dTTP was present in the triphosphates in the nucleus the relative amounts of all four triphosphates in the nucleus increased and dTTP appeared to be present only in the nucleus. The total concentration of deoxyribonucleoside triphosphates was calculated to be 3 x 10-4 m in the nucleus and 9 x 10-5 m in the cytoplasm. During inhibition of DNA synthesis by hydroxyurea, the nuclear content of dGTP was reduced to about 10 % and that of dATP to about 20 %, suggesting that the inhibition of DNA synthesis was primarily due to a depletion of the dGTP pool. Inhibition of DNA synthesis by thymidine resulted in greatly increased nuclear pools of dTTP, dGTP, and dATP and in a disappearance of the nuclear dCTP. Inhibition of DNA synthesis is explained by the absence of the dCTP pool. During thymidine inhibition, nuclear dTTP concentration was calculated to be about 3 x 10-3 m, as compared with 2 x 10-4 m in the cytoplasm. In most cases we thus found an unequal distribution of deoxyribonucleoside triphosphates between the cytoplasm and the nucleus. The mechanism responsible for this effect appears to discriminate among the four deoxyribonucleoside triphosphates.