In order to better understand the bioleaching mechanism, expression of genes involved in energy conservation and community structure of free and attached acidophilic bacteria in chalcopyrite bioleaching were investigated. Using quantitative real-time PCR, we studied the expression of genes involved in energy conservation in free and attachedAcidithiobacillus ferrooxidansduring bioleaching of chalcopyrite. Sulfur oxidation genes of attachedA. ferrooxidanswere up-regulated while ferrous iron oxidation genes were down-regulated compared with freeA. ferrooxidansin the solution. The up-regulation may be induced by elemental sulfur on the mineral surface. This conclusion was supported by the results ofHPLCanalysis. Sulfur-oxidizingAcidithiobacillus thiooxidansand ferrous-oxidizingLeptospirillum ferrooxidanswere the members of the mixed culture in chalcopyrite bioleaching. Study of the community structure of free and attached bacteria showed thatA. thiooxidansdominated the attached bacteria whileL. ferrooxidansdominated the free bacteria. With respect to available energy sources during bioleaching of chalcopyrite, sulfur-oxidizers tend to be on the mineral surfaces whereas ferrous iron-oxidizers tend to be suspended in the aqueous phase. Taken together, these results indicate that the main role of attached acidophilic bacteria was to oxidize elemental sulfur and dissolution of chalcopyrite involved chiefly an indirect bioleaching mechanism.