In order to clarify the interrelationship between prolactin and progesterone on normal mammary gland growth, SHN strain of virgin mice were given the pituitary grafting under the kidney capsule, the subcutaneous implantation of progesterone pellet or the bilateral ovariectomy singly or in combination at 40 days of age. All mice were killed 60 days after treatments and mammary end-bud formation, mammary DNA content and plasma prolactin level were examined. The single pituitary grafting elevated plasma prolactin levels in both the intact and the ovariectomized animals, however, mammary gland growth was enhanced only in the former. Progesterone pellet implantation also stimulated mammary gland growth in the intact mice, while prolactin changed little. Progesterone effect was nullified by ovariectomy associated with the decline in plasma prolactin level. The simultaneous single pituitary grafting and progesterone pellet implantation in combination or the grafting with three pituitaries restored mammary gland regression by ovariectomy. All findings have demonstrated that the manifestation of progesterone effect on mammary glands is dependent upon prolactin and that the role of prolactin is both direct on the glands and indirect through its luteotropic effect - through its stimulation of ovarian progesterone secretion.