We investigated the conductance of pea thylakoid membranes and their capacity for photophosphorylation as function of the extraction of chloroplast coupling factor CF1. The degree of extraction was varied via the incubation time in EDTA-containing hypo-osmolar medium and was measured by rocket electroimmunodiffusion. The conductance of thylakoid membranes was measured by flash kinetic spectrophotometry. The time course of extraction followed the time course of thylakoid swelling. Contrary to expectation increasing loss of CF1 did not primarily increase the velocity of proton efflux from each vesicle. Instead proton-tight vesicles were converted to leaky ones, which lost phosphorylating activity. Two subpopulations occurred, although both types of vesicles, leaky and proton-tight ones, were CF1-depleted to a similar degree. This implied that only a small fraction of CF1-lacking CF0 was functional as a proton channel. Tight vesicles had no functional channels while leaky ones had at least one. We determined the proportion of tight vesicles in three independent ways: via the residual phosphorylation activity, via measurements of proton efflux and via measurements of the electric relaxation across the membrane. The results obtained were identical. A statistical evaluation of the data led us to the following conclusions. EDTA treatment produced vesicles containing approximately 10(5) chlorophyll molecules, equivalent to a total of approximately 100 CF0CF1 per vesicle. Even at the highest degree of extraction (75% of total CF1 extracted) only 2.5 out of 75 exposed CF0 per vesicle were proton-conducting. The unit conductance of one open CF0 channel was 169 +/- 18 fS at pH 7.5 and room temperature. At an electrical driving force of 100 mV this was equivalent to the passage of approximately 10(5) protons/s. The most important consequence of this relatively high unit conductance was that a single open CF0 channel was capable of dissipating the protonmotive force of one vesicle, thereby deactivating the whole remaining catalytic capacity of this vesicle.