The structure of purified histone H5 in 1 M-NaClO4 and of H5 in nuclei was analysed by digestion with either one of three endoproteinases, papain, subtilisin or elastase, which preferentially cleave unstructured protein regions (and additionally with trypsin). Digestion with papain and subtilisin produced 'limiting' resistant peptides (p1 and s1) that contain the central region between residues 18-20 and residue 114. Digestion of purified H5 with elastase generated resistant peptides e1 and e2, and that of H5 in nuclei, peptide e2. Peptides e1 and e2 contain the region from residues 22 to 114 and 109 respectively. These results show that a central region of H5 encompassing the sequence between residues 18-22 and residue 114 is folded into a compact structure. A central structured 'core' domain ranging from residues 22 to 100 is defined by the limit trypsin peptide t3, which is identical to the previously described fragment GH5 [Aviles et al. (1978) Eur. J. Biochem. 88, 363-371]. Generation of peptides e2 and t3, as well as of resistant peptides of lower abundance, shows that the sites near Lys-100 and Lys-109 exhibit some proteolytic sensitivity, which may result either from an exposed location or from a locally less compact conformation. Significantly, all these structural features of H5 are manifested in the purified form as well as in nuclei. A role of the structured region from residues 101 to 114 for the interaction with linker DNA and the determination of its path is discussed.