X-ray structure analysis of the noncatalytic sites of F1-ATPase revealed that residue α-Asp261 lies close to the Mg of bound Mg-5'-adenylyl-β- γ-imidodiphosphate. Here, the mutation αD261N was generated in Escherichia coli and combined with the αR365W mutation, allowing nucleotide binding at F1 noncatalytic sites to be specifically monitored by tryptophan fluorescence spectroscopy. Purified αD261N/αR365W F1-ATPase showed catalytic activity similar to wild-type. An important feature was that, without any resort to nucleotide-depletion procedures, the noncatalytic sites in purified native enzyme were already empty. Binding studies with MgATP, MgADP, and the corresponding free nucleotides led to the following conclusions. Residue α-Asp261 interacts with the Mg of Mg-nucleotide in noncatalytic sites and provides a largo component of the binding energy (~3 kcal/mol). It is the primary determinant of the preference of noncatalytic sites for Mg-nucleotide. The natural ligands at these sites in wild-type enzyme are the Mg-nucleotides and free nucleotides bind poorly. Under conditions where noncatalytic sites were empty, αD261N/αR365W F1 showed significant hydrolysis of MgATP. This establishes unequivocally that occupancy of noncatalytic sites by nucleotide is not required for catalysis.