The presence in At. ferrooxidans of canonical copper resistance determinants does not explain the extremely high copper concentrations this microorganism is able to tolerate. This suggests that At. ferrooxidans may have additional copper resistance mechanisms. New possible copper resistance determinants were searched by using 2D-PAGE and real time PCR (qRT-PCR). Results showed the up-regulation of RND-type Cus systems and different RND-type efflux pumps in At. ferrooxidans grown in the presence of copper, suggesting that these proteins may be implied in resistance to this metal. Furthermore, the up-regulation of putative periplasmatic disulfide isomerases was also seen in the presence of copper. These proteins are most likely involved in the formation and rearrangement of disulfide bonds in proteins in the periplasm. Copper ions catalyze the formation of incorrect disulfide bonds in proteins. However, the up-regulated disulfide isomerases found could restore native disufide bonds allowing cell survival. In conclusion, At. ferrooxidans may resist high copper concentrations by using additional copper resistance strategies in which cell envelope proteins are very important. This knowledge could be used to select the best fit members of the bioleaching community to attain more efficient industrial biomining processes.