The isolation of mammalian sperm heads from their tails is complicated by the relatively high density of the tails, but facilitated by the fact that protamine condensation of the sperm chromatin and the insolubility of the perinuclear theca make the sperm nucleus stable in sodium dodecyl sulfate. Two methods are described for the isolation of rodent sperm nuclei using sucrose step gradients in which the sperm nuclei are only centrifuged one time, minimizing potential damage by mechanical stress.