NEMALINE myopathy, a congenital neuromuscular disease, is one of several muscle disorders in which apparently abnormal Z lines, or Z line-type structures emanating from Z lines, has been described1–6. There has been considerable speculation concerning the chemical composition and structural arrangement of proteins in nemaline rods2,4,7–12, but these features have remained unclear: part of this uncertainty is related to lack of understanding of the intact Z line. The only Z line constituent for which there is substantial evidence is α-actinin13–16. We have described the preparation and properties of a Ca2+-activated neutral protease (termed CAF) from muscle that is highly specific in its activity towards myofibrillar proteins and structure17,18. Addition of CAF to isolated myofibrils or to teased muscle fibrils releases undegraded α-actinin from the Z line, with no noticeable effect on myosin, actin, or the remaining myofibrillar structure. We report here the use of CAF as a dissection tool to strip away the dense, amorphous component of the nemaline rods, exposing an underlying set of longitudinal filaments running parallel to the long axis of the original rod. Decoration of these filaments with heavy meromyosin19 shows that the longitudinal filaments of nemaline rods are composed of actin.