Three fundamentally different chloroplast ATP synthase samples of increasing complexity were visualized by atomic force microscopy. The samples are distinguishable in respect to the isolation technique, the detergent employed, and the final subunit composition. The homo-oligomer III was isolated following SDS treatment of ATP synthase, the proton-turbine III+IV was obtained by blue-native electrophoresis, and complete CFO was isolated by anion exchange chromatography of NaSCN splitted ATP synthase. In all three ATP synthase subcomplexes 14 and only 14 circularly arranged subunits III composed the intact transmembrane rotor. Therefore, 14 protomers built the membrane-resident proton turbine. The observed stoichiometry of 14 is not a biochemical artifact or affected by natural growth variations of the spinach, as previously suggested. A correlation between the presence of subunit IV in the imaged sample and the appearance of a central protrusion in the narrower orifice of the oligomeric cylinder III14 has been observed. In contrast to current predictions, in chloroplast FO the subunit IV can be found inside the cylinder III14 and not at its periphery, at least in the reconstituted 2D arrays imaged.