The possible occurrence of water-soluble vitamin D sulfate in fresh mammalian milk was studied. Water-soluble vitamin D sulfate was isolated as its water-insoluble Ba salt from the aqueous layer of fresh deproteinized milk. After repeating careful analyses, about 204 I.U. [International Units] of vitamin D was shown to be present in 1 liter of fresh cow''s milk in a water-soluble form; about 36 I. U. per liter in a lipid-soluble form. In the case of fresh human milk, it was estimated to be about 950 I.U. per liter in a water-soluble form while 15.7 I. U. in a lipid-soluble form.