We have used high-resolution electron spectroscopic imaging to study the ultrastructure of negatively stained chicken gizzard vinculin, A careful examination of uranium salt-stained molecules revealed a high versatility of the overall shape of vinculin, for which an element of high flexibility is mainly responsible, This neck region links the vinculin head, probably consisting of the biochemically defined 90-kDa N-terminal fragment, to the rod-like tail. The hinge allows for sharp kinks in the molecule, so that head and tail structures can contact each other, By electron spectroscopic imaging, we were able to reveal substructural components in both head and tail regions, The head resembles a cloverleaf-like structure, consisting of three globular centers of mass, surrounding a protein-deficient center in a planar arrangement and of a short, stem-like fragment, The tail contains four spherical protein masses arranged like pearls on a string, Our data reveal a substructural organization of vinculin which is consistent with its presumed function as a structural component of microfilament attachment sites and support the concept of cryptic ligand-binding domains, previously based on biochemical evidence. (C) 1996 Academic Press, Inc.