The translocase of the outer mitochondrial membrane TOM is responsible for the transport of all nuclear-encoded proteins into mitochondria. In this study, the determination of subunit composition, mass, and stoichiometry of TOM core complex gives hints about the mode and strength of interaction between single subunits. The main constituent of the translocase TOM is the channel-forming Tom40. Here, the recombinant expression, purification, and folding of two human Tom40 isoforms is described. Secondary structure analyses revealed a dominant beta-sheet structure and a small alpha-helical content in connection with a high thermal stability. To increase the stability of human Tom40, the potential energetic contribution of the predicted beta-strands was calculated and three rather unstable beta-strands in the transmembrane domain were substituted by hydrophobic amino acids. Thermal stability and solvent denaturation revealed a significant stabilization of the modified Tom40.