Commercially available calf thymus DNA could be fractionated into different populations by DEAE-cellulose column chromatography, presumably due to the tertiary folding of the solid fibrous form of DNA which opened up gradually in solution. Depending on the duration in solution or the concentration of the solution, various peaks could be obtained by eluting the DEAE column with 0.01 M sodium phosphate buffer at pH 7.0 and 0.01, 0.4, 0.5, 0.6 and 1.0 M NaCl in the same buffer. The DNA fraction in each peak gave very narrow bands on 1% agarose gel electrophoresis, probably reflecting the folding and thus shielding of charges rather than molecular weight. The present fractionation procedure might be useful in characterizing the complicated network structure of calf thymus DNA and in studying the unfolding of DNA tertiary structure.