MgADP in combination with fluoroscandium (ScFx) is shown to form a potently inhibitory, tightly bound, noncovalent complex at the catalytic sites of F1-ATPase. The F1·MgADP·ScFx complex mimics a catalytic transition state. Notably, ScFx caused large enhancement of MgADP binding affinity at both catalytic sites 1 and 2, with little effect at site 3. These results indicate that sites 1 and 2 may form a transition state conformation. A new direct optical probe of F1-ATPase catalytic transition state conformation is also reported, namely, substantial enhancement of fluorescence emission of residue β-Trp-148 observed upon binding of MgADP·ScFx or MgIDP·ScFx. Using this fluorescence signal, titrations were performed with MgIDP·ScFx which demonstrated that catalytic sites 1 and 2 can both form a transition state conformation but site 3 cannot. Supporting data were obtained using MgIDP-fluoroaluminate. Current models of the MgATP hydrolysis mechanism uniformly make the assumption that only one catalytic site hydrolyzes MgATP at any one time. The fluorometal analogues demonstrate that two sites have the capability to form the transition state simultaneously.