Abstract Sonicated calf thymus DNA was fractionated by rate zonal centrifugation into seven fractions with weight average molecular weights ranging from 0.28 to 1.3 × 10 6 daltons, as determined by sedimentation equilibrium and light scattering measurements (the latter are described in the accompanying paper). Electron microscopy and sedimentation equilibrium analysis revealed these fractions to be narrowly disperse with M w/ M n ratios averaging about 1.06. Intrinsic viscosities and sedimentation rates were measured and found to vary linearly with molecular weight in double-logarithmic plots in fair agreement with previously published functions relating these parameters for low molecular weight DNA. The average value for β from the Mandelkern— Flory equation was 2.59 × l0 6, also agreeing with reported estimates of this parameter for short DNA. These data will be used in the second paper of this series to calculate the persistence length of the DNA fragments in each of the seven fractions by light scattering and hydrodynamic theories for the Kratky—Porod worm-like coil.