The three-dimensional structure of the recombinant histone rHMfB from Methanothermus fervidus, an archaeon that grows optimally at 83°C, has been determined by nuclear magnetic resonance methods. This is only the third structure of a protein from a hyperthermophilic organism (optimal growth at temperatures above 80°C). Signal assignments were made using a combination of homonuclear-correlated, 15N-double resonance and 15N, 13C triple resonance NMR experiments. Long range dipolar interactions for the symmetric homodimer were identified from two-dimensional 13C- double half-filtered and three-dimensional 13C-filtered NMR data obtained for a heterolabeled-dimer. A family of 33 structures was calculated using DSPACE TMwith a total of 609 NOE-derived interproton distance restraints, including 22 intraresidue, 192 sequential, 300 medium-range (two to five residues), 86 long-range intramolecular (more than five residues) and 112 intermolecular distance restraints. The monomer subunits consist of three α-helices, extending from residues Pro4 to Ala15 (helix I), Ser21 to Ala50 (helix II) and Lys56 to Lys68 (helix III), as well as two short segments of β-strand comprised of residues Arg19 to Ser21 and Thr54 to Ile55. Helices I, II and III contain N-terminal capping boxes, and helices I and II contain C-terminal caps. The structure of the (rHMfB) 2dimer appears very similar to the dimer subunits within the histone core octamer of the chicken nucleosome. The presence of a canonical “histone fold” motif in rHMfB is consistent with the HMf family of archaeal histones and the eukaryal nucleosome core histones having evolved from a common ancestor. The (rHMfB) 2dimer contains several structural features that may impart thermal stability (or non-lability), including two novel hydrophobic “proline Ncaps”, four interhelical hydrogen bonds and short N- and C-terminal disordered tails.