Thermoanaerobacter tengcongensis, one of many thermophilic organisms, survives harsh living conditions in temperatures ranging from 50 to 80 degrees C. In this comprehensive analysis, we present a robust approach, 2-DE and MALDI-TOF MS, to compare and identify the bacterial proteins responding to the temperature stress. In total, 164 spots of 2-DE were found with the significant changes in spot volume at three culture temperatures, 55, 75, and 80 degrees C, respectively; furthermore, 87 unique proteins were characterized by MS. Our results reveal that the electrophoretic images of the bacterial proteins, extracted from two culture temperatures (55 and 75 degrees C), had similar patterns; however, the bacteria cultured at 80 degrees C had dramatically decreased their spot volumes. Additionally, the temperature-sensitive proteins are broadly divided into two groups: specific expression at certain temperatures and consistent changes of expression responsive to temperature. For instance, three proteins closely related with redox regulation, dihydrolipoamide acyltransferase, NADH:ubiquinone oxidoreductase, and ferredoxin, were only detected in the bacteria cultured at 55 degrees C. Whereas, two chaperonins, GroES and GroEL, were found to show a consistent increase during the elevated temperatures with the determinations, either by MS or Western blot. The proteomic information, thus expedites our understanding of the molecular mechanisms regarding how thermophilic bacteria adapt to the alterations in living environment.