Proton pumping in thylakoid membranes and backflow of protons through the active ATP synthase CFO-CF1 (where CFO is the proton channel and CF1 is the catalytic portion) were investigated by flash spectrophotometry. A steady pH difference across the membrane was generated by continuous measuring light, supplemented by voltage transients that were generated by flashing light. In the presence of Pi and ADP, the electric potential transients elicited transients of proton flow via CFO-CF1, typically 1.3 H+ per CF1 and flash group. Proton flow was blocked by N,N'-dicyclohexylcarbodiimide, acting on the channel component CFO, and tentoxin, acting on the catalytic component CF1.The half-rise time was 40 ms in '1120 and 78 ms in 2H20. ATP synthesis under conditions of flashing light and transient proton flow was characterized by a Km(Pi) of only 14 ,uM, contrasting with a Km of several hundred micromolar for continuous AtP synthesis at high rate. This might reflect a resistance to Pi dhffusion. The degree of proton delocalization in the chemiosmotic coupling between redox reactions and ATP synthesis is under debate. In thylakoids, it has been proposed that intramembrane proton buffering domains act as ducts for protons between pumps and ATP synthases. In this work, transient proton flow by way of CFO-CF1 was completely tracked from the lumen, across the membrane, and into the suspending medium. Proton uptake from the lumen and charge flow across the membrane occurred synchronously and in stoichiometric proportion. The uptake of protons from the lumen by CFO-CF1, half completed in 40 ms, was preceded by release of protons from water oxidation into the lumen, half completed in <1 ms. Hence, pumps and ATP synthases were coupled through the lumen without involvement of intramembrane domains.The chemiosmotic theory of oxidative and of photophosphorylation places proton pumps and proton translocating ATP synthases in a membrane and couples them by lateral proton flow through two aqueous bulk phases at different electrochemical potentials of the proton (1, 2). Though supported by a vast body of evidence, the delocalized coupling concept has been challenged from its origin (3). In one class of alternative models, proton pumps and ATP synthases are thought to be coupled in pairs rather than sharing common bulk phases (localized coupling or microchemiosmotic concepts, e.g., ref. 4). In another class, proton flow is thought to be contained in the membrane (e.g., ref. 5) or confined to the membrane/ water interface (e.g., refs. 6 and 7), and the protons in these localized domains are supposed not to be equilibrated with adjacent aqueous phases. The bulk of the information interpreted to favor localized coupling concepts was obtained using mitochondria. It resulted from an evaluation of the energetics of phosphorylation or from a comparison of the rates of stationary electron flow and of ATP synthesis in the presence of inhibitors to the former and the latter (reviewed in refs. 4 and 8). Unambiguous conclusions were not reac...