Publisher Summary The nine outer dense fibers that surround the length of the axoneme in a mammalian sperm are joined anteriorly to the connecting piece, and differ from one another in cross-sectional shape and size. Fibers 1, 2, 4, 5, 6, 7, and 9 extend longitudinally from the connecting piece to almost the end of the principal piece. Fibers 3 and 8 terminate at the limit between the midpiece and the principal piece. The dense fiber polypeptides are unrelated to contractile proteins. In rat sperm the dense fibers account for about 40% of the total sperm proteins, indicating that during spermiogenesis, the synthesis and assembly of these fibrillar structures should be one of the most important events. To learn more about the function and morphogenesis of the dense fibers, a method to isolate these structures from sperm was developed that has been described in this chapter. The chapter details the solutions needed, the process of sperm isolation, isolation of the outer dense fibers, and purification of the dense fiber polypeptides. Rat epididymal sperm were obtained from up to 30 Holtzman rats, and mouse epididymal sperm from a Rockefeller strain. The sperm are allowed to diffuse into the solution. Fresh human ejaculates were obtained from healthy donors and were selected for a large proportion of normal cells and more than 30 million sperm. The dissociation of the sperm structures is followed by phase microscopy until the only visible structures are the sperm heads and the complex of outer dense fibers. Treatment of human and mouse sperm with solution A also produced the complex of dense fibers. With rat, mouse, and bull sperm presence of 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and 0.2 mM N,N’-tetraacetic in the solutions prevent proteolysis. During the isolation of human sperm dense fibers, a degradation of the polypeptides was observed.