Histones H2b and H3, phosphorylated in vitro with the catalytic subunit of protein kinase I from rabbit skeletal muscle, were used to estimate the influence of histone phosphorylation upon histone-histone complex formation. Stoichiometry and interaction affinity of the complexes H2a-H2b, H4-H2b, and H4-H3 were determined by using the continuous variation method based on circular dichorism or fluorescence intensity. All complexes exhibit a 1:1 stoichiometry in sodium phosphate or sodium chloride solution of pH 7.0. The association constants of the complexes containing phosphorylated H2b were only slightly reduced, whereas that with phosphorylated H3 was strongly reduced relative to those of the nonphosphorylated species.