F1-ATPase is an ATP-driven rotary motor in which a rod-shaped gamma subunit rotates inside a cylinder made of alpha3beta3 subunits. To elucidate the conformations of rotating F1, we measured fluorescence resonance energy transfer (FRET) between a donor on one of the three betas and an acceptor on gamma in single F1 molecules. The yield of FRET changed stepwise at low ATP concentrations, reflecting the stepwise rotation of gamma. In the ATP-waiting state, the FRET yields indicated a gamma position approximately 40 degrees counterclockwise (= direction of rotation) from that in the crystal structures of mitochondrial F1, suggesting that the crystal structures mimic a metastable state before product release.