Nuclei of rat ventral prostate have been demonstrated to possess a protein phosphatase activity utilizing 32P-labelled, lysine-rich histone (calf thymus) as the phosphoprotein substrate. This phosphatase has a pH optimum of 7.1 and was stimulated by the sulfhydryl protective agents dithiothreitol and 2-mercaptoethanol. This nuclear protein phosphatase did not appear to require divalent cations; rather, small inhibitions of activity were found in the presence of 2.4 mM Mg2+, Mn2+, and Ca2+. Divalent cations such as Zn2+ or Cu2+ were found to be much stronger inhibitors, giving about 80% inhibition at 1 mM. Monovalent cations were also found to inhibit the histone phosphatase, e.g., 43% at 200 mM NaCl. Ammonium molybdate did not influence the enzyme activity whereas ADP and ATP reduced it by 72 and 82% respectively at 1 mM. There was no change in activity of the histone phosphatase up to 96 h post-orchiectomy when specific activity was based per unit of nuclear protein. However, a small decrease is noted if specific activity is expressed per unit of nuclear DNA (19% at 48 h and 36% at 96 h orchiectomy). This difference reflects the decreased nuclear protein content of the prostate observed following castration. Our data suggest that the decline in prostatic nuclear histone phosphorylation observed following orchiectomy is not due to increased phosphatase activity.