Nebulin is a family of giant sarcomere matrix proteins of 600-900 kDa in most vertebrate skeletal muscles. Recent sequence analysis suggests that human nebulin is mainly composed of a large number (greater than 200) of conserved repeats of approximately 35 residues. Two cloned nebulin fragments, consisting of 6 and 8 of the repeats, have been expressed in E. coli using the pET3d vector. Both F-actin cosedimentation and solid-phase binding assays demonstrated a specific binding of these nebulin fragments to actin. This finding suggests that nebulin is a giant protein which binds actin at multiple sites in a template-manner. The presence of an actin-binding template protein in the skeletal muscle sarcomere may have significant implications in the assembly and function of the contractile apparatus.