Thermus thermophilus HB8 is a model microorganism for industrial applications because of its thermophilic enzymes, and for basic bacteriology to understand the coordination of the biological functions of the genome-encoded enzymes at the cellular level. Here, we present 2DE reference maps of T. thermophilus HB8 in the pH ranges 4–7 and 6–11 obtained with whole-cell lysates. PMF analysis using MALDI-TOF-MS and MS/MS analysis using nano-scale LC and quadrupole TOF-MS identified 258 different proteins among the 306 protein spots on 2DE gels. Functional classification indicated that 56%, 16%, and 14% of the identified proteins were related to metabolism, genetic information process, and cellular process, respectively. Detailed classification of the metabolism-related proteins suggested that during the exponential phase, amino acid and carbohydrate metabolism are major metabolic processes, whereas nucleotide and lipid metabolism are minor ones. On the other hand, volume quantification analysis revealed that proteins involved in the translational process, nucleotide metabolism, and central carbon metabolism were most abundantly expressed in the exponential phase.