The relationship between the ultrastructure and protein components of Z-disks was studied using psoas muscles of rabbit and breast muscles of chicken. Glycerinated fiber bundles of these muscles were treated with a solution containing 0.1 mM Ca2+ to induce a structural weakening of Z-disks non-enzymatically. During the treatment, clear geometrical configurations of Z-filaments could be observed along with removal of amorphous matrix under an electron microscope. Even after a prolonged treatment for 14 d in which all Z-disks were entirely weakened, entangled Z-filaments were left in the original region of the disks. Immunoelectron microscopic observations showed that antibodies against alpha-actinin bound to the entangled Z-filaments, forming dense lines at the position of the original Z-disks. On SDS-PAGE of Z-disk substances, alpha-actinin remained unchanged and Mr 75,000 and 55,000 proteins were removed during the Ca2+-treatment. We therefore conclude that alpha-actinin is a component of Z-filaments, and that the amorphous matrix is composed at least of these Mr 75,000 and 55,000 proteins.