Mouse DNA was fractionated by preparative centrifugation in density gradients of Cs2SO4 containing 3,6-bis(acetatomercurimethyl)dioxane (BAMD). The effects of temperature, BAMD/nucleotide molar ratio and solvent on the fractionation were explored. The fractions so obtained were investigated by analytical centrifugation in CsCl density gradient and by hybridization with a number of gene probes. These approaches led (a) to the definition of satisfactory conditions for the rapid fractionation of mouse DNA; (b) to the localization of a number of genes in mouse DNA fractions; and (c) to a better understanding of the mosaic organization of the mouse genome and, more specifically, to a better estimate of both the intermolecular and intramolecular compositional heterogeneity of mouse DNA in the (75–150) × 103-base size range.