Comparisons were made between the metabolic activities of whole mitochondria and intact mitochondrial inner membrane preparations from magnesium-deficient and control rats in a basic medium without exogenous magnesium. Magnesium deficiency partially uncoupled oxidative phosphorylation in whole mitochondria and completely uncoupled it with inner membrane preparations. Addition of 1 mM MgCl2 to the medium prevented the total uncoupling of inner membranes and gave ADP:O values similar to those obtained with whole mitochondria from magnesium-deficient rats. No impairment in proton extrusion by intact inner membranes or in ATPase activity by either intact or fragmented inner membranes was detected during magnesium deficiency, but there was evidence of increased membrane permeability to the inward movement of protons. It is concluded that magnesium deficiency probably increases the permeability of the mitochondrial inner membrane and this weakens the coupling between oxidation and phosphorylation.