Histone phosphokinase activity is present in nucleoli and chromatin isolated from nuclei of rat ventral prostate. The activity (utilizing lysine-rich histone as substrate) in each of these subnuclear fractions is maximal at pH 8.0-8.2, is optimally stimulated in the presence of dithiothreitol and NaCl, and requires Mg2+. Enzymic properties described for the histone phosphokinase activities are similar for nucleolus and chromatin, but are different from those demonstrated by kinases which readily phosphorylate phosvitin. Histone phosphokinase activity of the nucleolus declines about 18% at 24 h and 45% at 48 h post orchiectomy. This decrease in activity is prevented by testosterone replacement therapy. Chromatin-associated histone phosphokinase activity also declines following orchiectomy (21% at 48 h and 66% by 96 h). Thus, the rates of decline of nueleolar and chromatin-associated histone phosphokinase activity in orchiectomized rats described here are slower compared to the enzymic activity toward acidic protein substrates. This further suggests that changes in androgenic status results in differential effects on the protein phosphokinase reactions associated with the prostatic nucleus.