The effect of the ATPase activity of Manduca sexta V₁ ATPase by the amphipathic detergent lauryldimethylamine oxide (LDAO) and the relationship of these activities to the subunit composition of V₁ were studied. The V₁ was highly activated in the presence of 0.04-0.06% LDAO combined with release of the subunits H, C, and F from the enzyme. Increase of LDAO concentration to 0.1-0.2% caused the characterized subcomplexes A₃B₃HEGF and A₃B₃EG with a remaining ATPase activity of 52 and 65%, respectively. The hydrolytic-active A₃B₃EG subcomplex has been visualized by electron microscopy showing six major masses of density in a pseudo-hexagonal arrangement surrounding a seventh mass. The compositions of the various subcomplexes and fragments of V₁ provide an organization of the subunits in the enzyme in the framework of the known three-dimensional reconstruction of the V₁ ATPase from M. sexta (Radermacher, M., Ruiz, T., Wieczorek, H., and Grüber, G. (2001) J. Struct. Biol. 135, 26-37).