A possible relationship between mitochondrial Mg2+ levels, structural configurations, and functional steady states has been studied in rat liver mitochondria. The results show that the concentration of mitochondrial Mg2+ in respiratory state 4 is definitely higher than in respiratory state 3. The metabolic transition from state 3 to state 4 and vice-versa is associated with reversible influx-efflux of about 10 nmol of Mg2+ per mg protein. The net uptake of this aliquot of Mg2+ is a necessary condition in order for the metabolic transition to state 4, both structurally and functionally, to occur. This process requires a threshold concentration of external Mg2+ greater than 5 mM. The phosphorylative mechanism does not appear to depend on the presence or absence of external Mg2+. The role of Mg2+ on the attainment and maintenance of the structural and functional steady state 4 seems to be correlated with its regulatory effect on the concentration of the mitochondrial P(i).