Inorganic phosphate is present in milk at a concentration that is several-fold higher than in maternal plasma. In cultured mammary tissues from 12-14 day pregnant mice, the intracellular concentration of ³²PO₄ was 6 times higher than in the culture medium after a 4 hour treatment with ³²PO₄. Of the principle lactogenic hormones (insulin (I), cortisol (H) and 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 TCA insoluble molecules. In a time-course study, the onset of the PRL stimulation of ³²PO₄ uptake and incorporation occurred 8-12 hours 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 V_max of PO₄ uptake was determined to be about 7.7 mMol/hr/L cell water; the apparent K_m was about 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.