Respiration-driven Mg2+ efflux from rat heart mitochondria has been studied in different conditions. Almost total release of Mg2+ from the mitochondria occurs upon addition of a proton/bivalent cation exchanger, A23187. The content of Mg2+ remaining in mitochondria after A23187 treatment is the same if part of the mitochondrial Mg2+ has already been extruded through the energy-linked mechanism. Some inhibition of Mg2+ efflux is observed in the presence of high concentrations of La3+ (100 micro M). A proton/monovalent cation exchanger, nigericin, completely prevents Mg2+ efflux, whereas a cation conductor, valinomycin, considerably stimulates it. The results indicate that the main part of mitochondrial Mg2+ is present in the membrane-bounded compartment, probably in the matrix space. The driving force of the Mg2+ efflux appears to be the proton gradient (deltapH) created by mitochondrial respiration.