Cyanogen bromide peptide analyses of cross-linked histones show that histone 2B possesses separate binding sites for histones 2A and 4. Various polymorphic forms of H2A and H4 in chromatin are bound to H2B through these binding sites. These interactions are thus a fundamental structural feature of chromatin. Additional data strongly suggest that H2B is bound to H2A and H4 simultaneously in vivo. Mouse L cells were treated with either tetranitromethane (TNM) or ultraviolet (uv) light at 280 nm in order to induce the cross-linking of histone 2B in their chromatin to either histone 4 or 2A. The characteristics of these cross-linking agents and the reactions they induce suggest that the cross-links define loci within the actual binding sites between these histones. Characterization of the cyanogen bromide peptide fragments of the H2B-H4 and H2B-H2A dimers showed that the C terminal half of H2B contains the link to H4 (also near its C terminus), whereas the N-terminal half contains the link to H2A. Treatment of the cells with both uv light and TNM results in the appearance of a single new trimer which we presume to be H2A-H2B-H4. We conclude form this that H2B interacts simultaneously with H2A and H4 in chromatin through separate binding sites. Further analysis shows that several acetylated subspecies of H4 are represented in the H2B-H4 dimer and that at least two of the polymorphic forms of H2A are present in the H2B-H2A dimer. These data reveal that the H2A-H2B-H4 association is an important underlying structural feature of chromatin.