The entire 2nd thoracic mammary gland of the immature virgin BALB/c mouse was stimulated to full lobulo-alveolar (LA) growth after 120 h organ culture in hormone supplemented medium. The minimal hormonal combination required was insulin (I) + prolactin (Prl) + aldosterone (A). The corticosteroid was replaceable by oestradiol-17beta (E) + progesterone (P). The combination I alone or I + the steroid hormone(s) failed to induce the LA development and similar results were also evident in presence of Prl + the steroids. Incubation of the glands in medium with I + Prl + A activated a sequential rise of RNA, protein and DNA synthesis. A near maximal increase of RNA synthesis was present at 48 h in the medium with I + Prl, addition of the steroid hormones did not show further stimulatory effect. Supplementation of the medium with I + Prl and the adrenal or the ovarian steroids was needed for maximal activation of protein synthesis at 72 h and DNA replication at 96 h. The medium with I alone did not show a substantial rise of macromolecular biosynthesis in the mammary gland in organ culture. The highest level of DNA polymerase activity was observed at 72 h in glands cultivated in medium with I + Prl and A or E + P. Only a modest increase of DNA polymerase activity was present in glands cultivated with I alone or I + Prl. Prior treatment of the glands (cultivated with I + Prl + A) with actinomycin D or puromycin resulted into 44 and 40% reduction of DNA polymerase activity suggesting hormone-induced synthesis of the enzyme before the rise of DNA synthesis in the mammary cells at 96 h in organ culture. Significance of these results with respect to the action of the "growth-promoting" hormones in the mammary gland in organ culture and in the animal has been discussed.