S phase CHO cells chronically exposed to 41.5 degrees C exhibited an apparent resistance to further cell killing after 4-6 h. During the treatment interval, cells were observed to progress out of S phase and into G2, mitosis, and the next cell cycle. Progression through S was delayed by about 2 h and an equivalent mitotic delay was observed. After entry into mitosis, heated cultures showed an altered nuclear morphology, presumably as the result of abnormal division occurring during the treatment. Tritium suicide experiments at this temperature showed that clonogenic, as well as nonclonogenic, cells progressed during this period. When S phase cultures were chronically heated at 42 degrees C, however, the delay in transit through S phase was 8-10 h, and an accumulation in G2 phase was observed. Therefore, our results show that, in S phase cultures heated at 42 degrees C, cell killing continues while cells progress through S, suggesting that chronic thermotolerance cannot be expressed in S phase. Furthermore, at 41.5 degrees C, cells which progress out of S during the treatment express resistance to heat killing in subsequent cell cycle phases. In summary, our results indicate that, although chronic tolerance is not expressed during heating in S phase, it is expressed after the cells progress out of S phase.