The roles and interdependence of DNA sequence and archaeal histone fold structure in determining archaeal nucleosome stability and positioning have been determined and quantitated. The presence of four tandem copies of TTTAAAGCCG in the polylinker region of pLITMUS28 resulted in a DNA molecule with increased affinity (delta,deltaG of ~700 cal.mol-1) for the archaeal histone HMfB relative to the polylinker sequence, and the dominant, quantitative contribution of the helical repeats of the dinucleotide TA to this increased affinity has been determined. The rotational and translational positioning of archaeal nucleosomes assembled on the (TTTAAAGCCG)4 sequence, and on DNA molecules selectively incorporated into archaeal nucleosomes by HMfB have been established. Alternating A/T- and G/C-rich regions were located where the minor and major grooves, respectively, sequentially face the archaeal nucleosome core, and identical positioning results were obtained using HMfA, a closely-related archaeal histone also from Methanothermus fervidus. However, HMfA did not have similarly high affinities for the HMfB-selected DNA molecules, and domain-swap experiments havshown that this difference in affinity is determined by residue differences in the C-terminal region of a-helix 3 of the histone fold, a region that is not expected to directly interact with DNA. Rather this region is thought to participate in forming the histone dimer:dimer interface at the center of an archaeal nucleosome histone tetramer core. If differences in this interface do result in archaeal histone cores with different sequence preferences, then the assembly of alternative archaeal nucleosome tetramer cores could provide an unanticipated and novel structural mechanism to regulate gene expression.