In vitamin D-deficient rats intestinal calcium transport increased significantly 4 hours after an injection of prolactin, reached a maximum after 8 hours, and declined to preinjection levels after 24 hours. Similarly, in vitamin D-deficient rats fed a diet low in calcium or phosphorus prolactin stimulated an increase in serum calcium in both groups and an increase in serum phosphorus in the rats fed the diet low in phosphorus. Thus it appears that prolactin affects organs involved in calcium regulation in a manner that is independent of the vitamin D endocrine system.