Ca2+-activated protease (CAF) digestion of glycerinated nemaline myopathy muscle removed the electron-dense material covering rods and Z-lines and exposed longitudinal backbone filaments, 6-7 nm wide, which span the lengths of the original rods. Decoration of the exposed filaments (which are responsible for the periodicity parallel to the long axis of intact nemaline rods) with heavy meromyosin (HMM) proved they are actin filaments. After CAF treatment, cross-striated periodical patterns in longitudinal sections and Z-filament-like proteins connecting actin filaments seen in cross-section disappeared. This suggests that alpha-actinin may be involved in formation of this pattern because of the specificity of CAF toward alpha-actinin. Gel electrophoresis of CAF-treated nemaline muscle showed that most alpha-actinin is released into the supernatant, whereas the residue is mainly actin and myosin. Electron microscope examination of longitudinal sections of intact rods shows an oblique filament pattern, thin (7 nm) lines, thick (11 nm) lines, and an amorphous-appearance previously observed in normal Z-lines, patterns observed depend on sectioning angle and section thickness. In cross-section, rods show small square net (SS) and basket-weave (BW) forms. The SS form predominates and coexistence of the 2 forms, which also occur in normal Z-lines, is observed. Results support the idea that rods are lateral polymers of Z-line units. We think that the length of rods, as well as the width of Z-lines, is determined by the amount of overlap of actin filaments of opposite polarity. Initiation of rod formation may be due to deregulation of actin filament length.