The purpose of this investigation was to study the response of the hematopoietic stem cell, spleen colony-forming unit (CFU-S), to hyperthermia. We have shown that CFU-S can acquire a transient resistance to further heating (thermotolerance). Hyperthermia was applied in vitro to nucleated bone marrow cells in McCoy's 5A medium plus 15% fetal bovine serum. Day-10 CFU-S (CFU-S10) were detected as spleen colonies after inoculation into the tail vein of irradiated (450 cGy plus 4 h plus 400 cGy) Balb/c male mice. Thermotolerance development was detected with a "step-up" heating protocol consisting of heating for various times at 42 degrees C followed immediately with a thermal challenge of 26 min at 44 degrees C. The inverse of the slopes of the heat "dose-response" curves (D degree +/- SE) of the normotolerant CFU-S heated to 42 degrees, 42.5 degrees, 43 degrees, 43.5 degrees, and 44 degrees C were 108 +/- 13, 54 +/- 8, 25 +/- 1, 17 +/- 2, and 12 +/- 5 min, respectively. A plot of the slopes of the heat "dose-response" relationships versus the inverse of the absolute temperature (Arrhenius plot) showed an inflection at approximately 43 degrees C. Analysis of the regression coefficient above and below the inflection point (Arrhenius analysis) yielded inactivation enthalpies (+/- SE) of 598 +/- 130 kJ/mol (143 +/- 31 kcal/mol) and 1205 +/- 171 kJ/mol (288 +/- 41 kcal/mol), respectively. The difference in inactivation enthalpy indicates a change in mechanism in the thermal inactivation of CFU-S above and below 43 degrees C, possibly due to thermotolerance development during exposure to temperatures less than 43 degrees C. Prolonged incubation at 42 degrees C for up to 180 min with a step-up to 44 degrees C for 26 min showed that CFU-S survival increased rapidly from 0.25 (26 min at 44 degrees C) to 0.52 within 10 min. The thermotolerance ratio (TTR, ratio of the surviving fraction of the maximum thermotolerant cells to that of the normotolerant cells) was 2.1. Both the higher inactivation enthalpy for exposures less than 43 degrees C and the rapid increase in survival during the "step-up" heating experiments at 42 degrees C demonstrate that CFU-S can develop thermotolerance during prolonged hyperthermia. These results suggest that thermotolerance can influence the thermal response of pluripotent bone marrow stem cells heated during whole-body or local-regional clinical hyperthermia protocols.