The effect of supraoptimal temperature on macronuclear DNA synthesis in Tetrahymena was studied by radioautography during prolonged heat and heat-shock synchronization treatments. Prolonged heat treatments (34 degrees C) delayed the initiation of S, but did not appreciably delay DNA synthesis in progress. Return to optimal temperature (28 degrees C) 50 or 100 min later resulted in initiation of S, in delayed cells, at a rate greater than in controls. During the synchronization treatment, most cells were unable to enter S during a heat shock, but initiated S with a slight delay during the following intershock period. These cells were not appreciably delayed in completion of S by subsequent heat shocks. Supraoptimal temperature appears to affect the DNA synthetic cycle near the G(1) to S transition. Cells subjected to the heat-shock treatment in early G(1) all participated in one S period, and many underwent a succession of two S periods. DNA synthesis occurred in about 50% of the cells between EST and the first synchronous division, with the likelihood of DNA synthesis becoming greater the longer the interval between these two events. In some cells no detectable DNA synthesis occurred between EST and the second synchronous division. It was concluded that a precise temporal alternation of DNA replication and cell division is not obligatory in Tetrahymena.