H1 histone was selectively extracted from calf thymus chromatin by treating with 0.6 M sodium chloride. The H1-depleted chromatin showed no activity for phosphorylation or dephosphorylation of H1 histone. Chromatin was reconstituted from the H1-depleted chromatin and H1 histone, which was extracted from calf thymus chromatin and purified after phosphorylation at serine 37 by cyclic AMP-dependent protein kinase. The rate of phosphorylation of H1 histone at serine 37 was estimated to be over 73%. The effects of phosphorylation of H1 at serine 37 in chromatin on structure and function of chromatin were studied using the reconstituted chromatin. When sodium chloride concentration in the reaction medium was increased from 0 to 80 mM, chromatin showed increased template activity and reduced nuclease sensitivity. The chromatin reconstituted from H1 and the H1-depleted chromatin showed reduced template activity and reduced nuclease sensitivity. The phosphorylation of H1 histone at serine 37 had no effect on template activity or sensitivity to micrococcal nuclease of the reconstituted chromatin.