To further our understanding of the biology of the thermophilic bacterium Geobacillus thermoleovorans T80, we now report the first proteomic analysis of the insoluble subproteome of this isolate. A combination of both shotgun and multidimensional methodologies were utilized, and a total of 8628 peptides was initially identified by automated MS/MS identification software. Curation of these peptides led to a final list of 184 positive protein identifications. The proteins from this insoluble subproteome were functionally classified, and physiochemical characterization was carried out. Of 15 hypothetical conserved proteins identified, we have assigned function to all but four. A total of 31 proteins were predicted to possess signal peptides. In silico investigation of these proteins allowed us to identify four of the five bacterial classes of signal peptide, namely, (i) twin-arginine translocation; (ii) Sec-type; (iii) lipoprotein, and (iv) ABC transport. In addition, a number of proteins were identified that are known to be involved in the transport of compatible solutes, known to be important in microbial stress responses.