The fate of ^3H-thymidine incorporated into newly synthesized DNA of mouse L cells was analyzed. Under conditions in which DNA synthesis proceeded continuously, the incorporated radioactivity was periodically lost and regained during a 135-min chase as judged by either labeling index, acid-insoluble counts per cell, or mean grain count. When DNA synthesis was inhibited by hydroxyurea, the periodic changes of the incorporated radioactivity were not observed. The periodical changes were regarded as the result of an actual metabolic changes in ^3H-labeled DNA of viable cells and the loss of radioactivity seems to require active continued DNA synthesis.