The predominant source of vitamin D is the synthesis of cholecalciferol in the skin by the action of sunlight; however, due to the relative lack of sunlight, the intake of vitamin D from food is emphasized during winter, especially in the northern countries. Only a few foods (fish, eggs, wild mushrooms, meat, and milk) are natural sources of vitamin D. In addition, the content of vitamin D in foods is generally low, and some groups of people obtain amounts of vitamin D that are too small from their diet. The present study was designed to determine whether it is possible to increase the vitamin D content of egg yolk by giving hens feed containing elevated levels of cholecalciferol. Three cholecalciferol levels were tested: 26.6 (1064), 62.4 (2496), and 216 microgram (8640 IU)/kg feed. Egg yolk samples were taken after 0, 4, 5, and 6 weeks and were assayed for the presence of cholecalciferol and 25-hydroxycholecalciferol using an HPLC method. According to the present study, there was strong positive correlation between cholecalciferol content in poultry feed and cholecalciferol (r = 0. 995) and 25-hydroxycholecalciferol (r = 0.941) content in egg yolk.