The molecular weight and purity of calf thymus DNA, prepared by different procedures of isolation, were determined. The highest molecular weight was obtained by the method of Blin and Stafford (12 x 10(7)). This DNA contained 17% protein. The further purification of this DNA led both to increase of purity and to reduction of the molecular weight. The relationship between molecular weight and achieved purity was found to be a monotonic sigmoid-shape function ranging between the molecular weights of 12 x 10(7) and 12 x 10(6) and between the purities of 17 and 0.7% protein. After achieving this highest level of purity (0.7% protein content), despite repetition of the purification cycles, no further increase of purity or reduction of the molecular weight was achieved. The use of [14C]T4 bacteriophage DNA as internal standard demonstrated that the reduction of the molecular weight does not result from an artifact, such as shear or enzymatic degradation, but that the high-molecular-weight calf thymus DNA preparation must be considered to consist of either aggregates of small subunits stabilized by laterally attached proteins or of tandemly joined units connected by protein or peptide linkers.