An in vitro DNA synthesizing system consisting os isolated nuclei from HeLa cells which had been treated with inhibitors of protein synthesis was investigated. Treatment with both 30 microgram/ml cycloheximide and 10 microgram/ml puromycin of S-phase cells reduced the rate of DNA synthesis immediately; however, the overall DNA synthesis continued for up to 4 h with a diminished rate and then ceased. In the nuclei which were isolated from the cells which had been incubated with these drugs for 6 h, little incorporation of [3H]TTP into acid-insoluble materials was observed. Addition of cytosol prepared from cells actively synthesizing DNA induced the incorporation of [3H]TTP in these nuclei, while little induction was observed by the addition of cytosol prepared from drug-treated cells in spite of the fact that the latter cytosol stimulated DNA synthesis in isolated nuclei from non-treated cells. The induced DNA synthesis was shown to require Mg2+, all four deoxyribonucleoside triphosphates and ATP, and to proceed discontinuously. The activity inducing DNA synthesis in drug-treated nuclei fluctuated with the phases in a cell cycle and it was not ascribed solely to DNA polymerase alpha nor to DNA ligase.