H+-driven FoF1-ATP synthase plays a major role in energy production in most organisms. It converts the electrochemical potential generated by the H+-gradient across membranes into the rotation of the c-subunit ring in Fo and then into that of the gamma-subunit in F1. The c-ring comprises 8–15 c-subunits, depending on the biological species. The crystal structures of the c-rings of H+-driven FoF1 in detergent micelles provided four different active site structures. In this work, membrane-embedded c-rings was analyzed by solid-state NMR. We used a wheat-germ extract system for cell-free expression of specifically isotope-labeled TFoc-rings. The most critical point in cell-free production is the formation of active rings from the synthesized c-subunit monomers. We examined this by comparing their structure and function with those of the c-rings prepared from TFoF1 complex. Since the active-site structures in crystals were different from one another, we focused our attention on this. To highlight the active site, SAIL(stereo-array-isotope-labeled)-Glu and Asn were used. We have successfully obtained structural information on the active site of the TFoc-rings in lipid membranes. The chemical shift of Calpha of Glu56 essential for H+-translocation revealed that its carboxyl group is protonated in membranes, forming the H+-locked conformation with Asn23. However, the interaction between the residues turned out to be weak. It will make the flipping out of the side chain of Glu56 easier on interaction with the adjacent H+-channel subunit, a. In contrast, the chemical nature of the essential acidic amino acid residue in E. coli Foc-ring was found to be different from that of TFoc-ring according to the chemical shift of the carboxylate.