We have found that histone H5 (or H1) induces physiological nucleosome spacings and extensive ordering on some plasmid constructions, but not on others, in a fully defined in vitro system. Plasmid pBR327 containing DNA insertions with lengths close to 300 base-pairs permitted histone H5 to induce a remarkable degree of nucleosome alignment. Seventeen multiples of a unit 210(+/- 4) base-pair repeat, covering the entire plasmid, were detected. Plasmid pBR327, not containing a DNA insert, permitted continuous alignment of only a few nucleosomes. These observations suggest that a necessary requirement in this system for histone H5 (or H1)-induced nucleosome alignment on small (less than 4 kb; 1 kb = 10(3) bases or base-pairs) circular plasmids may be that the total DNA length must be close to an integer multiple of the nucleosome repeat length generated, a type of boundary effect. Consistent with this hypothesis, five deletion constructs of pBR327 (not containing inserts), that spanned 64% of the plasmid, and possessed DNA lengths close to integer multiples of 210 base-pairs, permitted nucleosome alignment by histone H5. We have also found that plasmid length adjustment is not a sufficient condition for nucleosome alignment. For example, plasmids pBR322 and pUC18 did not permit nucleosome alignment when adjusted to near-integer multiples of 210 base-pairs. Also, for pBR327 that contained a length-adjusted deletion in one particular region, appreciable nucleosome alignment no longer occurred. These data suggest that a contiguous approximately 800 base-pair region of pBR327, interrupted in pBR322 and not present in pUC18, can nucleate histone H5-induced nucleosome alignment, which can then spread to adjacent chromatin. Supporting this idea, a positioned five-nucleosome array appears to originate in the required region. Additionally, on a larger (6.9 kb) plasmid construction, the "chromatin organizing region" of pBR327 and adjacent DNA on one side of it exhibited preferred H5-induced nucleosome alignment.