Abstract Calf thymus chromatin is dissociated in 3 m-NaCl, 0.05 m-NaHSO 3 and 6 m-urea, pH 7.8. The presence of NaHSO 3 is necessary to prevent degradation of calf thymus histones. The protein is returned to the DNA by a stepwise dialysis into solutions of lower NaCl concentrations, maintaining constant urea and NaHSO 3 concentrations. At a given NaCl concentration, free protein is separated from DNA-bound protein on a Bio-Gel A50 column equilibrated in the same salt and urea concentrations as the dialyzed sample. Both free and DNA-bound histones are analyzed by analytical disc-acrylamide gel electrophoresis. Urea lowers the affinity of histones for DNA. None of the histones return to the DNA until a NaCl concentration of 0.2 m has been reached. The first histone to bind is lysine-rich histone I. The other histones return to the DNA during dialysis from 0.15 m-NaCl, 0.05 m-NaHSO 3 and 6 m-urea to 6 m-urea, which is followed by a final dialysis into 2 × 10 −4 m-EDTA, pH 6.2. When native chromatin is extracted with NaCl in the presence of 6 m-urea and 0.04 m-NaHSO 3, slightly lysine-rich histones IIb 1 and IIb 2 are removed at 0.15 m-NaCl, lysine-rich histone I at 0.3 m-NaCl and the arginine-rich histones III and IV at 0.6 m-NaCl. Increasing the urea concentration to 7 m lowers the NaCl concentration required to remove histones III and IV to 0.15 m-NaCl, but does not change the NaCl concentration required to remove histones I, IIb 1 and IIb 2. The NaCl concentration at which histones III and IV associate with DNA appears to be a function of the urea concentration and time used for extraction.