Nuclei isolated from rat ventral prostate rapidly incorporate 32P from [γ-32P]-ATP into phosphoproteins in vitro. The rate of phosphorylation with different preparations was 25.6 ± 1.94 (S.D.) nmoles 32P incorporated/mg of protein per h. Optimal phosphorylation was observed in the following reaction medium: Mg2+ (5-6 mM), ATP (2-4 mM) and Na+ (115 mM), at pH between 7.4 and 7.6 at 38°. Mg2+ was essential for the phosphorylation whereas divalent cations such as Zn2+, Mn2+, Cu2+, and Ca2+ were inhibitory when added in the presence of Mg2+. In the absence of Mg2+, only Ca2+ and Mn2+ could substitute partially for Mg2+ in the phosphorylation reaction. Of the various monovalent cations tested, highest rates of incorporation were observed with Na+ and only slightly less with K+ or NH4. In the absence of monovalent cations, or in the presence of Li+, considerable reductions in the phosphorylation were observed. Nucleoside triphosphates other than ATP could dilute the entry of 32P from [γ-32P]ATP to varying degrees. The phosphorylation of nuclear proteins was inhibited by p-mercuribenzoate and N-ethylmaleimide. Ouabain and 2,4-dinitrophenol had no effect. Sonication resulted in some loss of the activity whereas repeated freezing and thawing produced no effect on the ability of nuclei to incorporate 32P into phosphoproteins. On acid hydrolysis, the phosphoproteins of prostate nuclei yielded O-phosphorylserine as the predominantly phosphorylated amino acid. Orchiectomy of rats caused a decline in the rate of incorporation of 32P into phosphoproteins of ventral prostate nuclei. This was completely prevented by administration of testosterone. The effect of a single injection of testosterone on the nuclear phosphoproteins could be demonstrated within 30 min after the injection, suggesting a possible role of nuclear phosphoproteins in gene action in rat ventral prostate in response to testosterone. The effects of testosterone were not apparent in vitro. Cyclic AMP did not stimulate the phosphorylation of proteins in nuclei from normal or castrated rats; instead, a small inhibition was observed.