Spermatozoa from several mammalian species have been dissected by chemical methods to yield free heads, tails with attached midpieces, and tails from which the mitochondrial components of the midpiece were removed. Mouse and rat spermatozoa were cleaved by brief treatment with trypsin to yield free heads and tails, while human, guinea pig, and rabbit spermatozoa were cleaved by trypsin only after incubation with 2-mercaptoethanol or dithiothreitol. Spermatozoa were also cleaved at the junction of the head and the tail by treatment with acid and base. Mitochondria were removed from intact spermatozoa or isolated tails by mechanical shear after treatment with 2-mercaptoethanol or dithiothreitol. The dissected components of spermatozoa were fractionated with good yield and high purity by density gradient centrifugation. Ultrastructural analysis indicates that proteolytic cleavage to yield separated heads and tails occurs at a specific location in the neck of the spermatozoon, leaving the basal plate attached to the head of the cell. In contrast, after acid cleavage the basal plate remains with the midpiece. Proteolytic treatment has no apparent effect on any other spermatozoan structures, whereas acid or base treatment results in damage to the plasma membrane, the acrosome, and other structures. The specificity of the proteolytic cleavage suggests that a particular protein or group of proteins may be responsible for the linkage between the sperm head and tail.