It has been proposed that C-terminal two ␣-helices of the ⑀ subunit of F 1 -ATPase can undergo conformational transition between retracted folded-hairpin form and extended form. Here, using F 1 from thermophilic Bacillus PS3, we monitored this transition in real time by fluorescence resonance energy transfer (FRET) between a donor dye and an acceptor dye attached to N terminus of the ␥ subunit and C terminus of the ⑀ subunit, respectively. High FRET (extended form) of F 1 turned to low FRET (retracted form) by ATP, which then reverted as ATP was hydrolyzed to ADP. 5-Adenyl-,␥-imidodiphosphate, ADP ؉ AlF 4 ؊ , ADP ؉ NaN 3 , and GTP also caused the retracted form, indicating that ATP binding to the catalytic  subunits induces the transition. The ATP-induced transition from high FRET to low FRET occurred in a similar time scale to the ATP-induced activation of ATPase from inhibition by the ⑀ subunit, although detailed kinetics were not the same. The transition became faster as temperature increased, but the extrapolated rate at 65°C (physiological temperature of Bacillus PS3) was still too slow to assign the transition as an obligate step in the catalytic turnover. Furthermore, binding affinity of ATP to the isolated ⑀ subunit was weakened as temperature increased, and the dissociation constant extrapolated to 65°C reached to 0.67 mM, a consistent value to assume that the ⑀ subunit acts as a sensor of ATP concentration in the cell.A rotary motor F 1 -ATPase (F 1 ) 2 is a water-soluble portion of F 0 F 1 -ATP synthase, which catalyzes ATP synthesis/hydrolysis coupled with a transmembrane proton translocation (1, 2). F 1 has a subunit structure of ␣ 3  3 ␥␦⑀; ␣ and  subunits have a non-catalytic and catalytic nucleotide binding sites, respectively; ␥ subunit rotates in the ␣ 3  3 ring; ␦ subunit connects the ring to the stator part of F 0 ; and ⑀ subunit rotates together with ␥ subunit as a body. The ⑀ subunit (ϳ14 kDa) has a regulatory function and consists of N-terminal -sandwitch and C-terminal two ␣-helices (3, 4).Previous structural studies of F 1 indicated two conformations of the ⑀ subunit with different arrangement of the two ␣-helices, that is, retracted folded-hairpin form and partly extended form (Fig. 1, A and B) (5-8). Cross-linking studies suggested the third conformation with fully extended ␣-helices 3 (Fig. 1C) (9). Biochemical data have indicated that the ⑀ subunit adopts the extended form in the absence of nucleotide or in the presence of ADP, in which ATPase activity is inhibited, and that ATP counteracts ADP by favoring the retracted form, which is a noninhibitory conformation (9). Thus, it appears that the regulatory function of the ⑀ subunit is dependent on the drastic conformational transition that is affected by nucleotide and other factors. However, previous studies have not provided kinetic information on how these dynamic conformational transitions occur in the enzyme at work. Fluorescence resonance energy transfer (FRET) is a powerful technique that enables us to probe conformational...