The effect of hyperthermia in the 41-50 degrees C range on the Na+,K+-ATPase of CHO cells has been investigated. Three separate activities of the enzyme, namely, ATP hydrolysis, K+ uptake, and binding of the specific inhibitor, ouabain, have been measured independently. The results can be summarized as follows: (1) The ouabain binding capacity of intact cells decreased with increasing temperature and with increasing time at any one temperature. (2) The loss of ouabain-sensitive K+ uptake after treating cells at 45 degrees C was very similar to the loss of ouabain binding capacity. In contrast, the ATP hydrolyzing activity was much more resistant to heat. (3) The ouabain binding capacity was more resistant to hyperthermia at 45 degrees C if the cells were given a prior exposure at 45 degrees C 12 hr earlier. In contrast, the ATP hydrolyzing activity did not display this tolerance to heat.