Mammary glands from second generation vitamin D-deficient mice and rats were examined for their ability to make the major milk proteins, casein and a-lactalbumin, both in vivo and in vitro. The glands from the rachitic animals were morphologically indistinguishable from those of age-matched controls. When placed in explant culture, glands from vitamin D-deficient mice and rats underwent DNA synthesis at a rate comparable to that of glands from the vitamin D-replete controls. However, the hormonally induced synthesis of casein and a-lactalbumin was significantly reduced in explants of glands from rachitic us. control animals. The reduction in caseinsynthesizing ability by mouse mammary gland explants was not reversed by the addition of 10-8 or 10-6 M 1,25-dihydroxycholecalciferol to the culture medium, but could be reversed by pretreating the vitamin D-deficient mice with the D metabolite in vivo for 10 days before the onset of culture. The decrease in milk protein synthesis in culture is paralleled in vivo by a decrease in milk protein content in the milk and lactating mammary glands of vitamin D-deficient mice and rats. Both in vivo and in vitro, it is the two highest mol wt caseins that are most affected by the lack of vitamin D in the diet. These data suggest that vitamin D does not play a fundamental role in growth and morphological development of the normal mammary gland, but, rather, it is important in maintenance of full hormonally induced functional differentiation of the mature gland.