Membrane vesicles were isolated from the obligate anaerobic bacterium Clostridium acetobutylicum. Beef heart mitochondrial cytochrome c oxidase was inserted in these membrane vesicles by membrane fusion by using the freeze-thaw sonication technique (A. J. M. Driessen, W. de Vrij, and W. N. Konings, Proc. Natl. Acad. Sci. USA 82:7555-7559, 1985) to accommodate them with a functional proton motive force-generating system. With ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine-cytochrome c as the electron donor, a proton motive force (delta p) of -80 to -120 mV was generated in these fused membranes. This delta p drove the accumulation of leucine and lysine up to 40- and 100-fold, respectively. High transport activities were observed in fused membranes containing Escherichia coli lipids, whereas the transport activities in fused membranes containing mainly soybean lipids or phosphatidylcholine were low. It is suggested that branched-chain amino acids and lysine were taken up by separate systems. The effects of the ionophores nigericin and valinomycin indicated that lysine and leucine were translocated in symport with a proton.