1. Calcium movement and proton pulse production by mitochondria have been studied using pH-and K+-sensitive glass electrodes and the Ca++ indicator murexide.2. Anaerobic treatment led to the release of free Ca++ into the medium, the quantity depending on the preparative procedure and the composition of the medium.3. The ejection of protons by anaerobic mitochondria given small additions of oxygen was accompanied by the uptake of approximately equal numbers of calcium ions. Addition of ethylene glycol bis (B-aminoethy1)-N,N'-tetraacetic acid (EGTA) reduced the proton pulses to about 40 Ol0 of their maximum size in KC1 medium, and to 5°/0 in a sucrose medium.4. Mitochondria treated with EGTA and valinomycin gave proton pulses that were accompanied by approximately equal uptakes of K+.5. Mitochondria kept anaerobic for 20 min and then aerated were found to be without respiratory control by ADP unless EGTA was added before aeration.6. It is concluded that the production of proton pulses by untreated mitochondria a t the anaerobic-aerobic transition occurs only under conditions where sufficient free Ca++ has leaked out to allow energy-dependent exchange between external Ca++ and internal protons.It has been shown that proton pulse production by mitochondria a t the anaerobic-aerobic transition occurs with strict H+/O ratios for different substrates [l-31. This has been used as evidence for the 'chemiosmotic' theory of energy coupling which states that proton production is directly involved with the synthesis of ATP in mitochondria by an anisotropic ATPase, the energy for ATP synthesis supposedly being derived from a membrane potential arising from the charge separation of H+ and OHacross the sided ATPase within the membrane. The object of the present work has been to investigate whether proton production by mitochondria a t the anaerobic-aerobic transition can be correlated to a respiration-driven Ca uptake, since it has been well established that the energised uptake of Ca++ gives rise to a stoichiometric release of protons [4-71. If Ca++ uptake is the cause of the proton pulse production described by Mitchell, the appearance of protons in the medium is not indicative of an electrostatic charging of the membrane capacity. Since only one positive charge appears externally in exchange for one of the two positive charges on the Ca++, there must be a generation of one internal anion or OH-for each H+ ejected.Unusuul Abbreviation. Ethylene glycol bisv-aminoethyl)-N,N'-tetraacetic acid, EGTA.The use of a Ca++ indicator, murexide, in conjunction with a dual-wavelength spectrophotometer, as described by Chance and Mela [S], permits sufficiently sensitive measurements of Ca++ levels outside the mitochondria to be made during the anaerobicaerobic transition.The production of protons by anaerobic mitochondria in the presence of EGTA and valinomycin has been described by Mitchell and Moyle, who first interpreted it in terms of membrane conductance phenomena [9]. However, they later [lo] gave evidence showing a K+/H+ exchange re...