Because of channels in the outer membrane of the mitochondria, the content of the intermembrane space is similar to that of the content of the cytoplasm.

As electrons move down the proteins in the electron transport chain, the electrons lose energy to bring H+ ions from the mitochondrial matrix into the intermembrane space. As a concentration gradient of hydrogen ions forms, a protein called ATP synthase harnesses the potential energy of these ions and starts chemiosmosis, where the H+ ions reenter the matrix via this enzyme bound to the cristae (folds of the inner membrane). ADP and a phosphate group are combined to form ATP.