The Z-line in each striated muscle has a precisely defined width that corresponds to muscle fiber type, and it can enlarge several fold in nemaline myopathy. To explore the mechanism(s) underlying Z-line width and structure maintenance, a series of sarcomeric-alpha-actinin mutants tagged with myc-epitope was transfected into cultured chick myotubes. By double-staining transfected myotubes with myc and myofibrillar protein antibodies, we found that alpha-actinin mutants with deletion of the region from the beginning of the fourth spectrin repeat to the start of the EF-hands resulted in expansion of Z-line width, often displayed a doublet staining pattern, and resulted in formation of nemaline-like bodies in older myotubes under fluorescence microscope. Yeast-two hybridization analysis demonstrated that this region was involved in vinculin binding, and for vinculin to bind alpha-actinin, residues 1-116 and 258-323 were required. Hence, we have defined a critical region of s-alpha-actinin that affects the width and integrity of the Z-line. This region is at least involved in the interaction with vinculin.