We studied the secondary structure of the C-terminal domains of the histone H1 subtypes H1 degrees (C-H1 degrees ) and H1t (C-H1t) in the presence of macromolecular crowding agents (Ficoll 70 and PEG 6000) by IR spectroscopy. The carboxyl-terminal domain has little structure in aqueous solution but became extensively folded in the presence of crowding agents. In 30% PEG, C-H1 degrees contained 19% alpha-helix, 28% beta-sheet, 16% turns, and 31% open loops. Similar proportions were observed in 30% Ficoll 70 and for C-H1t in both crowding agents. The proportions of secondary structure motifs were comparable to those of the DNA-bound domain. Kratky plots of the small-angle x-ray scattering showed that in crowding agents the C-terminus had the compaction of a globular state. Progressive dissipation of the secondary structure and a linear increase in partial heat capacity with temperature together with increased binding of ANS indicated that the C-terminus is not cooperatively folded in crowded conditions. Native-like secondary structure and compactness in absence of folding cooperativity indicate that the C-terminus in crowding agents is in a molten globule state. Folding of the C-terminus in crowded conditions may increase the rate of the transition toward the DNA-bound state and facilitate H1 diffusion inside cell nuclei.