Previous studies have compared the relative free energies for histone octamer binding to various DNA sequences; however, no reports of the equilibrium binding affinity of the octamer for unique sequences have been presented. It has been shown that nucleosome core particles (NCPs) dissociate into free DNA and histone octamers (or free histones) on dilution without generation of stable intermediates. Dissociation is reversible, and an equilibrium distribution of NCPs and DNA is rapidly attained. Under low ionic strength conditions (<400 mM NaCl), NCP dissociation obeys the law of mass action, making it possible to calculate apparent equilibrium dissociation constants (K(d)s) for NCPs reconstituted on defined DNA sequences. We have used two DNA sequences that have previously served as model systems for nucleosome reconstitution studies, human alpha-satellite DNA and Lytechinus variegatus 5S DNA, and find that the octamer exhibits K(d)s of 0.03 and 0.06 nM, respectively, for these sequences at 50 mM NaCl. These DNAs form NCPs that are approximately 2 kcal/mol more stable than total NCPs isolated from cellular chromatin. As for mixed-sequence NCPs, increasing ionic strength or temperature promotes dissociation. van't Hoff plots of K(a)s versus temperature reveal that the difference in binding free energy for alpha-satellite and 5S NCPs compared to bulk NCPs is due almost entirely to a more favorable entropic component for NCPs formed on the unique sequences compared to mixed-sequence NCPs. Additionally, we address the contribution of the amino-terminal tail domains of histones H3 and H4 to octamer affinity through the use of recombinant tailless histones.