The mechanism of Bax-dependent cytochrome c release is still controversial and may also depend on the actual localisation of cytochrome C: (i) we studied the distribution of cytochrome c in sub-fractions of rat kidney mitochondria and found that 10-20% of the total cytochrome c was associated at the peripheral inner membrane and to some extent organised in the contact sites. (ii) Cytochrome c concentrations in the contact site fractions varied related to surface bound hexokinase activity. It decreased upon reduction of contact sites by glycerol or specific dissociation of the VDAC-ANT complexes by bongkrekate, whereas it increased upon induction of contacts by dextran or association of VDAC-ANT complexes by atractyloside. (iii) The outer membrane pore (VDAC) acquires high capacity for hexokinase binding by interacting with the ANT. Thus, surface-attached hexokinase protein indicated the frequency of VDAC-ANT complexes and the correlation between hexokinase activity and cytochrome c suggested association of the latter to the complexes. (iv) Substances affecting exclusively the structure of either hexokinase (glucose-6P) or cytochrome c (borate) led to a decrease only of the effected protein without changing the concentration of other contact site constituents. (v) Hexokinase was furthermore used as a tool to isolate the contact site forming complex of outer membrane VDAC and inner membrane ANT from Triton-dissolved membranes. Cytochrome c remained attached to the hexokinase VDAC-ANT complexes that were reconstituted in phospholipid vesicles. (vi) The vesicles were loaded with malate and BaxDeltaC released the endogenous cytochrome c from the reconstituted complexes without forming unspecific pores for malate. BaxDeltaC targeted a cytochrome c fraction associated at the VDAC-ANT complex. The cytochrome c organisation was dependent on the actual structure of VDAC and ANT. Thus, the BaxDeltaC effect was suppressed either by hexokinase utilising glucose and ATP or by bongkrekic acid both influencing the pore and ANT structure.