Rat ventral prostate nuclei were separated into three major fractions by mild digestion with micrococcal nuclease and two fractions by extensive digestion. All fractions contained androgen-binding sites. Almost 50% of nuclear binding sites were resistant to enzymic digestion when only 5-15% of total DNA was resistant. Under milder digestion conditions, 21% of nuclear binding sites were associated with an intermediate fraction, representing 16% of total nuclear DNA, which was enriched in specific androgen-regulated gene sequences. This fraction was rapidly degraded by more extensive digestion. The nuclease sensitivity of these particular genes was markedly influenced by castration and the administration of dihydrotestosterone to castrated animals. The nuclear content of both nuclease-resistant and -sensitive androgen-binding sites was decreased by castration. Whereas the administration of androgen to animals castrated 1 day previously preferentially replenished nuclease-resistant sites, nuclease-sensitive sites, including those associated with transcriptionally active regions, had apparent priority when androgen was supplied to animals castrated 7 days previously. The significance of these observations to the regulation of nuclear processes and the possible interrelationships of nuclease-sensitive and -insensitive sites are discussed.