The response of V ₁ ATPase of the tobacco hornwormManduca sexta to Mg ²⁺ and nucleotide binding in the presence of the enhancer methanol has been studied by CuCl ₂-induced disulfide formation, fluorescence spectroscopy, and small-angle X-ray scattering. When the V ₁ complex was supplemented with CuCl ₂ nucleotide- dependence of A-B-E and A-B-E-D cross-linking products was observed in absence of nucleotides and presence of MgADP+Pi but not when MgAMP·PNP or MgADP were added. A zero-length cross-linking product of subunits D and E was formed, supporting their close proximity in the V ₁ complex. The catalytic subunit A was reacted with N-44-7-(dimethylamino)-4-methylcoumarin-3-ylmaleimide (CM) and spectral shifts and changes in fluorescence intensity were detected upon addition of MgAMP·PNP, -ATP, -ADP+Pi, or -ADP. Differences in the fluorescence emission of these nucleotide-binding states were monitored using the intrinsic tryptophan fluorescence. The structural composition of the V ₁ ATPase from M. sexta and conformational alterations in this enzyme due to Mg ²⁺ and nucleotide binding are discussed on the basis of these and previous observations.