The Escherichia coli F1 ATPase, ECF1, has been examined by cryoelectron microscopy after reaction with Fab' fragments generated from monoclonal antibodies to the alpha and epsilon subunits. The enzyme-antibody complexes appeared triangular due to the superposition of three anti-alpha Fab' fragments on alternating densities of the hexagonally arranged alpha and beta subunits. The Fab' to the epsilon subunit superimposed on a beta subunit. A density was observed near the center of the structure in the internal cavity. The position of this central density with respect to peripheral sites was not fixed. Sorting of images of ECF1 labeled with the combination of three anti-alpha Fab' fragments plus an Fab' directed to the epsilon subunit gave three classes in each of which the central density was closest to a different beta subunit. The distribution of the central density among the three classes was measured for different ligand-binding conditions. When ATP was present in catalytic sites under conditions where there was no enzyme turnover (i.e., without Mg2+ present), there were approximately equal numbers of images in each of three classes. When ATP and Mg2+ were added and ATP hydrolysis was allowed to proceed, almost two-thirds of the images were in the class in which the central density was closest to the beta subunit superimposed by the epsilon subunit. We conclude that domains within the ECF1 structure, either the central mass or a domain including the epsilon subunit, move in the enzyme in response to ligand binding. We suggest that this movement is involved in coupling catalytic sites to the proton channel in the F0 part of the ATP synthase.