Previously, we identified the rat uterine nuclear type II [3H]estradiol binding site as histone H4 and an unknown 35 kDa protein with histone H4 immunoreactivity. Studies using calf thymus histones indicated that the 35 kDa protein was likely a dimer of histone H3 and H4. Further study of the type II site required methodology for producing sufficient quantities of recombinant histones, which retained ligand-binding properties. A variety of production methods produce sufficient quantities of histone for binding analyses were evaluated prior to finding a successful technique. The present studies describe techniques for the production of recombinant histones that retain the ligand binding properties of type II binding site. Binding studies with recombinant protein mirrored [3H]estradiol binding assays with rat uterine nuclear preparations. Histone H4 specifically binds [3H]estradiol with a low affinity (Kd approximately 20 nM) and in a cooperative fashion (curvilinear Scatchard plot; Hill coefficient approximately 4). Although histone H3 does not appear to bind ligand, regeneration of the histone H3/H4 pair produced a 35 kDa protein equivalent to the 35 kDa protein labeled with [3H]luteolin in rat uterine nuclear extracts and calf thymus histones. These data confirm the identification of histone H4 as a key component of the type II site. Future studies with recombinant proteins will lead to the identification of the "nucleosomal ligand-binding domain" for methyl-p-hydroxyphenyllactate (MeHPLA) and related ligands and delineation of their epigenetic control of gene expression and cell proliferation.