1. The addition of chelators to a suspension of mitochondria in a low-cation medium containing 9-aminoacridine caused a decrease in 9-aminoacridine fluorescence. The chelators removed bivalent cations from the membranes and allowed more 9-aminoacridine to move into the diffuse layer. The relative effect of EGTA and EDTA on the fluorescence suggested that the mitochondria are isolated with about equal amounts of Ca2+ and Mg2+ on the membranes. 2. The removal of the bivalent ions by chelators resulted in the inhibition of NADH oxidation. The inhibition could not be removed by adding sufficient decamethylenebistrimethylammonium ion (DM2+) to screen the fixed charges on the membranes and restore the fluorescence of 9-aminoacridine. This observation suggests that bivalent metal ions have a specific role in the oxidation of NADH. 3. Ca2+ and not Mg2+ reversed the inhibition of NADH oxidation caused by EGTA, whereas both reversed the inhibition caused by EDTA. This suggests that Ca2+ plays a specific role and that Mg2+ reverses the inhibition caused by EDTA by displacing the bound calcium from the chelator. 4. The results are interpreted as showing that Ca2+ plays a specific role in the oxidation of external NADH in addition to its ability to screen electrostatically or bind to the fixed charges associated with the surface of the membrane.