1. The interaction of electron-transporting particles from heavy mitochondria of ox heart with several fluorescent probes was examined. 2. 1-Anilinonaphthalene-8-sulphonate and 2-(N-methylanilino)naphthalene-6-sulphonate both show an energy-dependent response. 3. Energy transfer between the electron-transporting particles and the dyes and the kinetics of the dye–particle interaction were studied in order to locate the binding regions in the membrane. 4. The energy-dependent probe responses were shown to be a result of changes in the quantum yield of fluorescence of the bound dyes together with increased binding of the dyes to the energized membrane. 5. Fluorescence lifetime measurements were also used to observe changes on energization. 6. A new type of probe was found in pyrene-3-sulphonate, which may be regarded as a ‘volume indicator’ for the internal membrane binding region, since it shows a concentration-dependent excimer fluorescence. 7. By comparing the responses of all these dyes when energized particles are uncoupled, a membrane transition with a time-constant of 2–3s is inferred.

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