The higher order organization of chromosomes in human and mouse sperm-cell nuclei has been visualized by fluorescence in situ hybridization. In mouse testicular sperm, centromeres form several linear higher order structures that are attached to a heterochromatic chromocenter at the center of the nucleus. Telomeres of the acrocentric mouse chromosomes are associated with either the heterochromatic center or the nuclear membrane. Whole chromosome domains are arranged parallel to the heterochromatic chromocenter and occasionally wrapped around it. We propose that constitutive heterochromatin serves as the nucleation point for a cell-type-specific organization of mouse sperm chromatin. In human mature sperm, individual chromosomes also appear as elongated territories. When human sperm nuclei are lysed with high salt and detergent, the normally condensed sperm chromatin unravels into linear arrays that exhibit a discrete nodular substructure (of < 300 nm diameter). These beads may represent a basic packaging unit of sperm chromatin. Larger superbead-like structures are also seen along the extended chromatin fibers and these could contain multiples of the basic packaging unit. These observations indicate that mammalian sperm nuclei have a highly defined nuclear architecture. The implications of an ordered organization of DNA in sperm are unknown, but it is possible that functional compartmentalization of the sperm-cell nucleus influences the initiation and regulation of paternal gene activity in the early embryo.