The mitochondrial sheath of mammalian spermatozoa is adherent to an underlying organized network of electron-dense material termed the submitochondrial reticulum (SMR). In this manuscript we further characterize the substructure of the SMR and the outer mitochondrial membrane and provide new information on their structural interaction. The SMR resists solubilization by detergent and once partially released from the midpiece of extracted spermatozoa, it appears in negatively stained preparations as a network of longitudinally oriented ribons of fibrillar material which are laterally interconnected. In detergent-extracted specimens the SMR remains attached to the outer mitochondrial membrane thereby suggesting a firm structural interaction. Negatively stained specimens also demonstrate that the outer mitochondrial membrane possesses a paracrystalline substructure and it is suggested that ordered arrays of membrane-associated proteins are involved in the structural interaction with the SMR. The potential roles of this cytoskeletal complex during spermiogenesis and in mature sperm are discussed.