The response of an extremely thermoacidophilic archaeon, Metallosphaera sedula (growth temperature range, 50 to 79°C; optimum temperature, 74°C; optimum pH, 2.0), to thermal stress was investigated by using a 10-liter continuous cultivation system. M. sedula, growing at 74°C, pH 2.0, and a dilution rate of 0.04 hr ؊1 , was subjected to both abrupt and gradual temperature shifts in continuous culture to determine the responses of cell density levels and protein synthesis patterns. An abrupt temperature shift from 74 to 79°C resulted in little, if any, changes in cell density and a small increase in total protein per cell. When the culture temperature was shifted further to 80.5°C, cell density dropped to below 5 ؋ 10 6 cells/ml from 10 8 cells/ml, leading to washout of the culture. Operation at this temperature and slightly higher temperatures, however, could be achieved by exposing the culture to thermal stress more gradually (0.5°C increments). As a result, stable operation could be maintained at temperatures of up to 81°C, and the washout temperature could be increased to 82.5°C. Continuous culture operation at 81°C for 100 h (stressed phase) led to an approximately sevenfold lower steady-state cell density than that observed for operation at or below 79°C. However, sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis (both one and two dimensional) revealed significantly higher levels (sixfold increase) of a 66-kDa stress response protein (MseHSP60), immunologically related to Thermophilic Factor 55 from Sulfolobus shibatae (