FoF1-ATP synthases couple proton translocation with the synthesis of ATP using two rotary motors within the enzyme. To monitor inter-subunit movements during catalysis, we selectively attached two fluorophores to the F 1 part, sulforhodamine B at one of three ß-subunits and Cy5 at the ?-subunit. Reassembly with F o parts embedded in liposomes yielded functional holoenzymes. Fluorescence resonance energy transfer (FRET) was investigated in photon bursts of freely diffusing liposomes with reconstituted ATP synthases using a confocal set-up for single-molecule detection. Incubation with AMPPNP resulted in stable intensity ratios within a burst and three different FRET efficiencies. Upon ATP addition, a repeating sequence of three distinct FRET efficiencies was observed, indicating the stepwise movement of the ?-subunit during ATP hydrolysis. With this single-molecule FRET approach we detected a stepwise rotation of the ?-subunit under conditions for ATP synthesis (i.e. energization of the proteoliposomes by an acid-base-transition). The direction of rotation is opposite to the direction observed during ATP hydrolysis.