We report the isolation and sequence of cDNA and genomic clones for one of the two large subunits of human neurofilament, NF-M. Analysis of the sequence has allowed us to investigate the structure of the carboxy-terminal tail of this protein, and to compare it to that of the small neurofilament as well as to other intermediate filaments. The carboxy-terminal region of the protein contains a 13 amino acid proline- and serine-rich sequence repeated six times in succession. Within each repeat unit are two smaller repeats of the sequence Lys-Ser-Pro-Val. The four amino acid repeat may represent a kinase recognition site in a region of the protein that is known to be highly phosphorylated. We also note the presence of an additional heptad repeat at the extreme carboxy terminus of the protein. This region of 60 amino acids may be involved in coiled-coil interactions similar to those that facilitate the filament formation in the rod region. The human gene contains only two introns. Their positions correspond to two of the three introns found in the small neurofilament of the mouse. Thus, two of the three neurofilament genes of mammals have similar structures which are quite different from those of the other intermediate filaments. This finding suggests a common origin of the neurofilament subunits, whose evolutionary relationship to other intermediate filament genes is uncertain.