The three-dimensional crystal structure of hen apo-ovotransferrin has been solved by molecular replacement and refined by simulated annealing and restrained least squares to a 3.0-Å resolution. The final model, which comprises 5312 protein atoms (residues 1 to 686) and 28 carbohydrate atoms (from two monosaccharides attached to Asn473), gives an R-factor of 0.231 for the 11,989 observed reflections between 20.0- and 3.0-Å resolution. In the structure, both empty iron binding clefts are in the open conformation, lending weight to the theory that Fe3+ binding or release in transferrin proceeds via a mechanism that involves domain opening and closure. Upon opening, the domains rotate essentially as rigid bodies. The two domains of the N-lobe rotate away from one another by 53°, whereas the C-lobe domains rotate away each another by 35°. These rotations take place about an axis that passes through the two β-strands, linking the domains. The domains of each lobe make different contacts with one another in the open and closed forms. These contacts form two interdomain interfaces on either side of the rotation axis, and domain opening or closing produces a see-saw motion between these two alternative close-packed interfaces. The interdomain disulfide bridge (Cys478-Cys671), found only in the C-lobe, may restrict domain opening but does not completely prevent it.