Scaffold-associated regions (SARs) are A + T-rich DNA regions of several hundred basepairs that are known to bind specifically to nuclear or metaphase scaffolds. Surprisingly, histone H1 specifically associates with SARs. Under conditions of high co-operativity, at input ratios of H1 to DNA up to 15% (w/w), histone H1 binds preferentially to those DNA molecules harboring a SAR, leaving the non-SAR fragments free. Our experiments identify SARs as cis-acting sequences that nucleate co-operative H1 assembly along the SAR into the flanking non-SAR DNA. Experiments with simple DNA polymers implicate homopolymeric oligo(dA) · oligo(dT) tracts in preferential histone H1 assembly. The homopolymer oligo(dA) · oligo(dT) is, above a critical length of 130 base-pairs, a highly specific nucleator of H1 assembly. SARs may control the conformation of chromatin domains via a regulated H1 assembly and set up the potential transcriptional repertoire of the cell.