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The distribution of the functional group of the pH indicator bromthymol blue across thc osmotic barrier component (or M phase) of the cristae membrane separating the inner from the outer aqueous phase of rat liver mitochondria has been measured by three partially independent methods. In mitochondria in State 5, under the usual conditions, only about one third of the tot.al bromthymol blue reacts in the inner phase. In State 3, in presence of a high concentration of valinomycin in a KC1 medium, approximately the same proportion of the bromthymol blue reacts in the inner phase as in State 5 ; but in State 4, practically all the bromthymol blue is expelled across the M phase and registers the pH of the outer phase.The bromthymol blue technique is incompetent to measure the pH of the inner phase of mitochondria in State 4; but a method is described that will permit the approximate quantitative measurement of the intramitochondrial pH from the light extinction of bromthymol blue in State 5 (or in State 3 in presence of valinomycin) provided that the distribution of the indicator and the internal and external buffering powers of the mitochondrial suspension are known.The expulsion of the bromthymol blue from the mitochondria in State 4 is attributable to the development of an electric potential across thc M phase.According t o Chance and collaborators bromthymol blue can be used to indicate the pH on the inside of the cristae nicmbrane of intact respiring mitochondria [I -4,7-91 because it is tightly bound at a site on the inner side of the membrane. It has also been observed that bromthymol blue is bound to the membrane of submitochondrial particles, of bacterial chromatophores and of chloroplast grana, and it has been deduced that it indicates the inside pH in these vesicular preparations [5-71. The bromthymol blue technique for thc measurement of internal or membrane pH has consequently begun to play an important part in discussions of proton movements in oxidative and photosynthetic phosphorylation systems (see [lo, ill), and while some minor criticisms have been voiced [I 1,121, the method has generally been accepted as a reliable new tool for following internal or membrane pH changes in particle preparations.Bromthymol blue (BTB) is a very amphipathic substance, the molecule having both water-seeking acidic groups, and lipid-seeking hydrocarbon groups, and it therefore seemed likely to us that it would tend to become adsorbed at the surface of the lipid phases of the particle preparations in such a way that the hydrophobic portion of the molecule would be in the lipid and the hydrophilic (ionisable) groups Non-Standard Abbreviations. Carbonylcyanide ptrifluoroinethoxy phenylhydraeone, CFCCP; bromthymol blue, BTB.would be in the aqueous phases. One obvious type of environment in which the BTR might be localised would be provided by the interfaces between the osmotic barrier or M phase of the cristae membrane and the inner and outer aqueous phases [13], as suggested by Fig. 1.Such considerations imply that one cou...

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