THE enzyme ATP synthase, or F-ATPase, is present in the membranes of bacteria, chloroplasts and mitochondria, Its structure is bipartite, with a proton-conducting, integral membrane portion, F-0, and a peripheral portion, F-1. Solubilized F-1 is composed of five different subunits, (alpha beta)(3) gamma delta epsilon, and is active as an ATPase(1,2). The function of F-ATPase is to couple proton translocation through F-0 with ATP synthesis in F-1 (ref. 3), Several fines of evidence support the spontaneous formation of ATP on F-1 (refs 4, 5) and its endergonic release(6) at cooperative and rotating (or at feast alternating) sites(7). The release of ATP at the expense of protonmotive force might involve mechanical energy transduction from F-0 into F-1 by rotation of the smaller subunits (mainly gamma) within (alpha beta)(3), the catalytic hexagon of F-1 as suggested by electron microscopy(8), by X-ray crystal structure analysis(9) and by the use of cleavable crosslinkers(10). Here we record an intersubunit rotation in real time in the functional enzyme by applying polarized absorption relaxation after photobleaching to immobilized F-1 with eosin-labelled gamma, We observe the rotation of gamma relative to immobilized (alpha beta)(3) in a timespan of 100 ms, compatible with the rate of ATP hydrolysis by immobilized F-1. Its angular range, which is of at least 200 degrees, favours a triple-site mechanism of catalysis(7,11), with gamma acting as a crankshaft in (alpha beta)(3). The rotation of gamma is blocked when ATP is substituted with its non-hydrolysable analogue AMP-PNP.