The observation in this laboratory that respiration and Sr2+ import were stimulated by the addition of 3-hydroxybutyrate to suspensions of N-ethylmaleimide-treated mitochondria respiring in state 6, after the addition of Sr2+, in a sucrose medium containing choline as substrate, led to the proposal by FEBS Lett. 84, 135-1401 that there is a Ca2+(Sr2+)-3-hydroxybutyrate sylnporter in rat liver mitochondria.However, experiments described in the present paper support a different interpretation. Under the conditions of the experiments by Moyle and Mitchell, the rate of respiration and the poise of Sr2+ accumulation are mainly limited, not by A&,+, but by lack of respiratory substrate. Even though N-ethylmaleimide is a potent inhibitor of 3-hydroxybutyrate dehydrogenase, we have found that, somewhat surprisingly, under the special conditions of these experiments, sufficient 3-hydroxybutyrate dehydrogenase activity remains available to account for the 3-hydroxybutyrate-dependent respiratory stimulation and Sr2 ' import.The view held generally is that Ca2+ import by respiring rat liver mitochondria occurs electrophoretically via a uniporter specific for the divalent cations Ca2+, SrZ+, Mn2+ and Ba2+ [l]. Physiological Ca2+ export (as opposed to efflux caused by the lytic action of high [Ca"] [2]) is thought to occur by an electroneutral 2H+/CaZ + exchange system that has relatively low activity compared to the uniporter [3] and is inhibited by Sr2+ [4] (for a recent review see [S]). Therefore, the distribution of Ca2 + across the inner mitochondrial membrane does not achieve thermodynamic equilibrium during in vitro experiments. Instead, a steady state is reached in which CaZ' cycles between the medium and the matrix, and the concentration of unbound Ca2 + outside the mitochondria ([Ca2 ' 1;) is approximately micromolar. If the [Ca2+]6 is raised above this level, the rate of import rises above the rate of export, and net import occurs. As a result, the [Ca2']\ falls and, hence, the rate of import falls until a new steady state is reached [5].In 1977 Moyle and Mitchell [6] questioned whether net import of Ca2+ (or Sr2+) by respiring mitochondria involved a Ca2+-uniporter. They measured the +H+ (acid)/+Ca ratio during net import of Ca2+ by respiring mitochondria, either by adding a pulse of oxygen to an anaerobic mitochondrial suspension, or by adding CaZ+ to an aerobic mitochondrial suspension in state 4. In the presence of N-ethylmaleimide (NEM; to prevent electroneutral symport of H + with phosphate on the phosphoric acid porter [7]) this ratio was