Nebulin, a giant myofibrillar protein with size variants from 700 to 900 kDa in various skeletal muscles, has been proposed to constitute a set of inextensible filaments anchored at the Z-line and coextensive with actin filaments. To elucidate the architectural organization of this fourth set of myofilaments in the skeletal muscle sarcomere, we performed immunoelectron microscopic localization of epitope profiles of a number of site-specific monoclonal antibodies against cloned human nebulin fragments of known sequence loci. Monoclonal antibody N113, which is directed to fragment ND8 at approximately 300 residues away from the C-terminus, labelled the edges of Z-lines in both human quadriceps muscle and rabbit psoas muscle. Monoclonal antibody N101, which is directed to fragment NB5 near the N-terminal side, is localized to a single locus at 0.89 micron from the Z-line in human quadriceps muscle and 0.80 micron from the Z-line in rabbit psoas muscle. Additionally, monoclonal antibody N109, which is directed to fragment NA3 on the carboxy side of the adjacent fragment NB5, is localized at 0.76 micron away from the Z-line in rabbit psoas muscle. This one-to-one correspondence between epitope loci and sequence loci demonstrates that a single nebulin polypeptide spans the length of the thin filament with its C-terminus anchored at the Z-line. The epitope spacings of site-specific antibodies are consistent with the notion that the nebulin filament is uniform in mass density along its length.(ABSTRACT TRUNCATED AT 250 WORDS)