Catalytic and noncatalytic nucleotide sites of the F1 sector of ATP synthase were characterized by tryptophan fluorescence techniques. Seven Trp residues inserted in varied microenvironments in the catalytic sites, and one in the noncatalytic sites, were studied in mutant F1 enzymes which were otherwise devoid of Trp. Parameters measured were fluorescence lifetimes and dynamic and static quenching by acrylamide in the absence or presence of nucleotide. The results indicated that the solution structures of the mutant enzymes were consistent with reported crystal structures. In enzyme with three empty noncatalytic sites, all sites were relatively inaccessible to acrylamide, indicating a closed conformation. In contrast, when the three catalytic sites were empty, they were relatively and equally accessible to acrylamide, indicating an open conformation. This was the case in the presence or absence of Mg2+. Residue β-Trp-331 has been extensively used previously to determine nucleotide binding parameters in F1. Results here showed that in βY331W mutant F1, each of the three β-Trp-331 residues has an unusually long fluorescence lifetime, confirming that each contributes equally to the overall fluorescence signal.