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Background: Clostridium thermocellum is a Gram-positive thermophilic anaerobic bacterium with the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. Identification and characterization of protein complexes in C. thermocellum are important toward understanding its metabolism and physiology. Results: A two dimensional blue native/SDS-PAGE procedure was developed to separate membrane protein complexes of C. thermocellum. Proteins spots were identified by MALDI-TOF/TOF Mass spectrometry. 24 proteins were identified representing 13 distinct protein complexes, including several putative intact complexes. Interestingly, subunits of both the F1-F0-ATP synthase and the V1-V0-ATP synthase were detected in the membrane sample, indicating C. thermocellum may use alternative mechanisms for ATP generation. Conclusion: Two dimensional blue native/SDS-PAGE was used to detect membrane protein complexes in C. thermocellum. More than a dozen putative protein complexes were identified, revealing the simultaneous expression of two sets of ATP synthase. The protocol developed in this work paves the way for further functional characterization of these protein complexes. Background Clostridium thermocellum is a Gram-positive thermophilic anaerobe capable of degrading cellulose and producing ethanol and hydrogen. These qualities render C. thermocellum potentially useful for the production of biofuel from biomass. The cellulytic activities of this organism were well studied, the corresponding enzymes were found to organize into a cell surfaced bound multienzyme complex, termed cellulosome [1]. The arrangement of the enzymatic subunits in the cellulosome complex, made possible by a scaffoldin subunit, promotes enhanced substrate binding and degradation. However, other parts of its cellular functions are not well understood. Recently, a genome scale metabolic model was constructed [2], which provides a good basis for the overall understanding of its metabolism. Since membrane is where many important physiological functions, such as energy generation, protein trafficking, and small molecule transport [3], take place, we focused on membrane protein complexes as a start point to identify unique features of C. thermocellum. Identification of protein complexes in C. thermocellum is an important step toward understanding cellular behavior at an integrative level.