A modified isolation procedure provides a homogeneous A₁-ATPase from the archaeon Methanosarcina mazei Gö1, containing the five subunits in stoichiometric amounts of A₃:B₃:C:D:F. A₁ obtained in this way was characterized by three-dimensional electron microscopy of single particles, resulting in the first three-dimensional reconstruction of an A₁-ATPase at a resolution of 3.2 nm. The A₁ consists of a headpiece of 10.2 nm in diameter and 10.8 nm in height, formed by the six elongated subunits A₃ and B₃. At the bottom of the A ₃B₃ complex, a stalk of 3.0 nm in length can be seen. The A₃B₃ domain surrounds a large cavity that extends throughout the length of the A₃B₃ barrel. A part of the stalk penetrates inside this cavity and is displaced toward an A-B-A triplet. To investigate further the topology of the stalk subunits C-F in A₁, cross-linking has been carried out by using dithiobis[sulfosuccinimidylpropionate] (DSP) and 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (EDC). In experiments where DSP was added the cross-linked products B-F, A_x-D, A-B-D, and A _x-B_x-D were formed. Subunits B-F, A-D, A-B-D, and A-B-C-D could be cross-linked by EDC. The subunit-subunit interaction in the presence of DSP was also studied as a function of nucleotide binding, demonstrating movements of subunits C, D, and F during ATP cleavage. Finally, the three-dimensional organization of this A₁ complex is discussed in terms of the relationship to the F₁- and V₁-ATPases at a resolution of 3.2 nm.