The structure and spatial disposition of the matrix elements of the rat spermatozoan flagellar axoneme were investigated by thin sectioning, negative staining, and image analysis techniques. In order to visualize axonemal components by negative staining, spermatozoa were demembranated with Triton X-100 and the flagellum was either frayed by mechanical agitation or actively disintegrated by adding 1 mM ATP to the pH 9.0 extraction solution. In the dissociated flagella each axonemal doublet microtubule remained adjoined to its associated outer dense fiber, and the central pair remained as an intact complex. Flagella extracted in the presence of ATP displayed a specific pattern of axonemal disintegration: doublet tubules-dense fibers numbers 4, 5, 6, and 7 were extruded in a proximal direction from the fibrous sheath, while numbers 8, 9, 1, 2, and 3 remained associated as a half cylinder together with the central pair in the fibrous sheath. Of the extruded fibers, doublet tubules numbers 5 and 6 always remained adjoined to each other suggesting the possibility of a structural linkage similar to that found in typical 9 + 2 flagella. The radial spokes are arranged along the doublet tubule in sets of three; the spoke sets show a center to center spacing of 96 nm. Within a set, two spokes (nos. 1 and 2) are spaced 32 nm center to center, while spokes 2 and 3 are spaced 24 nm center to center. The polarity of the spoke triplets is such that the 32-nm spoke space is directed toward the anterior of the flagellum. An additional axonemal structure associated with the doublet tubule is located between spokes 1 and 2; this globular component lies close to the tubule surface and successive globules are spaced 96 nm center to center. Fine filamentous linkages extending between adjacent outer dense fiber-doublet tubule complexes were found to approach and join the doublet tubule at the level of the globule or spoke number 1; successive filamentous linkages are spaced at 96-nm intervals. The relationship of these components to the circumferential nexins is discussed. In negatively stained preparations, central pair microtubule complexes were frequently found which appeared to retain periodic "sheath" components. Optical transforms of negatively stained central pairs show a regular set of layer lines indexing on a fundamental 32-nm repeat. By masking selected areas of the specimen it is shown that the 32-nm reflection arises from only one member of the central pair and that in negatively stained images and optically filtered reconstructions the 32-nm periodicity arises from a set of "barb-shaped" structures which originate from the lateral edge of one central pair microtubule. The "barb-shaped" projections are pointed toward the proximal end of the flagellum and are attached along the tubule nearly in register with alternating "bridges" and "projections," both of which are spaced axially at 16 nm along the central pair complex. Filtering of images where the central pair retains its structural integrity with a doublet microtubule provides evidence of structural interactions between the 32-nm periodic sheath components and the radial spokes. The implications of these structural findings to flagellar motility are discussed.