Mitochondrial adenosine triphosphate (ATP) synthase comprises a membrane embedded F-o motor that rotates to drive ATP synthesis in the F-1 subunit. We used single-particle cryo-electron microscopy (cryo-EM) to obtain structures of the full complex in a lipid bilayer in the absence or presence of the inhibitor oligomycin at 3.6-and 3.8-angstrom resolution, respectively. To limit conformational heterogeneity, we locked the rotor in a single conformation by fusing the F6 subunit of the stator with the delta subunit of the rotor. Assembly of the enzyme with the F6-delta fusion caused a twisting of the rotor and a 9 degrees rotation of the F-o c(10)-ring in the direction of ATP synthesis, relative to the structure of isolated F-o. Our cryo-EM structures show how F-1 and F-o are coupled, give insight into the proton translocation pathway, and show how oligomycin blocks ATP synthesis.