Inorganic phosphate is present in milk at a concentration that is severalfold higher than in maternal plasma. In cultured mammary tissues from 12- to 14-day-pregnant mice, the intracellular concentration of ³²PO₄ was six times higher than in the culture medium after a 4-h treatment with ³²PO₄. Of the principal lactogenic hormones [insulin (I), cortisol (H), and prolactin (PRL)], only I and PRL (in the presence of H and I) stimulated ³²PO₄ uptake into cultured mammary tissues; H, by itself or in the presence of I or PRL, inhibited ³²PO₄ uptake. All three lactogenic hormones together effected the greatest stimulation of ³²PO₄ uptake. Similar hormone effects were observed with regard to ³²PO₄ incorporation into lipids and trichloroacetic acid-insoluble molecules. In a time course study, the onset of the PRL stimulation of ³²PO₄ uptake and incorporation occurred 8-12 h after PRL addition; in dose-response studies, the PRL effect was manifested with PRL concentrations of 50 ng/ml and above. From kinetic studies, the apparent maximal velocity of PO₄ uptake was determined to be ~7.7 mM · h¹ · l cell water¹; the apparent Michaelis-Menten constant was ~3-5 mM. The PRL effect on ³²PO₄ uptake was abolished when sodium was absent from the uptake medium. These studies thus demonstrate a complex interaction of three hormones (I, H, and PRL) in the regulation of ³²PO₄ uptake and incorporation into macromolecules in cultured mouse mammary tissues.