Here we describe the organization of the archaeal nucleosome, in which four archaeal histones are circumscribed by approximately 80 bp of DNA. Through a combination of sequence comparisons, 3D structural studies, site-directed mutagenesis and assays for DNA binding, we have assigned functions to most of the individual residues in the histone fold of the representative archaeal histone rHMfB. By SELEX selection, the sequences of DNA molecules that are most readily bound and wrapped by rHMfB into archaeal nucleosomes in vitro have been identified, and these define DNA structures that position archaeal nucleosome assembly.