Hen erythrocyte chromatin was treated with trypsin immobilized on collagen membranes and the unfolding of chromatin fiber was followed by light scattering at 90 degrees and flow linear dichroism. Chromatin was found almost completely decondensed when the bulk of H1 and H5 was digested while H3 was still intact. Further digestion leading to degradation of both H3 and the rest of H1 and H5 accounted for no more than 10-15% of the total effect. When chromatin with trypsin-cleaved H1 and H5 was titrated with increasing amounts of spermidine it folded similarly to the control sample. This finding suggests that charge neutralization appears a likely mechanism for maintaining the structure of the 30 nm chromatin fiber by the C-terminal domain of H1 and H5.