The magnitude of thermotolerance and the level of heat shock protein (HSP) expression have been measured in Chinese hamster ovary cells after gradual temperature transients from 37 degrees or 39 degrees to 42 degrees or 43 degrees C. When the level of thermotolerance was measured by clonogenic survival after challenging temperatures between 42 degrees and 43 degrees, substantial thermotolerance was observed. However, when the challenging temperature was raised to 45 degrees C, proportionally less thermotolerance was apparent. Heat shock proteins were quantitated by scanning densitometry of radiographs and, in the case of HSP 70, by immunoassay. Scanning densitometry revealed that low levels of heat shock proteins were synthesized during the heating gradients, but less than after a heat shock at 45 degrees C that delivered an equivalent heat dose. The immunoassay of HSP 70 levels measures both pre-existing and newly synthesized protein, and showed that there was net increase in HSP 70 during two of the heating gradients tested, despite the increase in synthesis noted on the gels. Higher turnover of HSP 70 at the elevated temperatures possibly accounted for the failure to detect a net gain in total protein. In contrast, the total amount of HSP 70 doubled during the 6 hr following a heat shock of 45 degrees for 10 min, an equivalent heat dose to one of the gradients where no net increase in HSP 70 was measured by immunoassay. It appears, then, that tolerance to hyperthemia at 43 degrees C or below may occur under some conditions in the absence of elevated levels of HSP 70, but tolerance to higher temperatures is more closely correlated with increased levels of heat shock proteins. However, even at higher temperatures, our data show disparities between the levels of HSP measured and the thermotolerance expressed.