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A study has been made of the metabolism of mammary tissue from midpregnant C3H mice in organ culture, with and without the hormones needed to induce lactogenesis in the tissue. Insulin greatly stimulated the uptake of glucose by the tissue, and the formation of labelled carbon dioxide and fatty acids from labelled glucose. The further addition of prolactin and corticosterone had little effect. Without hormones the rates of incorporation of [³H]thymidine into DNA and of [¹⁴C]uridine into RNA decreased during culture. Insulin stimulated these rates of incorporation and the further addition of prolactin stimulated them still more. Corticosterone had little effect. The rate of incorporation of [³²P]orthophosphate into casein fell during culture without added hormones, while insulin, or insulin plus corticosterone, had little effect on it. Prolactin, however, caused a very marked stimulation in cultures with insulin, which was only slightly increased by the further addition of corticosterone. The stimulation of ³²P incorporation due to prolactin was only evident after 12 hr. of culture. Delay in addition of prolactin by 12 hr. did not affect the response. Actinomycin D, cycloheximide and colchicine prevented the action of prolactin; hydroxyurea partly prevented it.