1. Seventeen 32-week-old White Leghorn laying hens were induced to become deficient in calciferol or in calcium (laying thin or soft shelled eggs) by withdrawing either cholecalciferol (27.5 micrograms/kg diet) or calcium (31 g/kg diet) supplements from the control diet. 2. The metabolic fate and metabolic clearance rate (MCR) of intravenously injected 3H-oestradiol-17 beta were then monitored for 40 min. 3. In both the calciferol and calcium-deficient groups a major oestrogen sulphate pathway was particularly affected, resulting in a decreased conversion of oestradiol-17 beta-3-sulphate to oestradiol-17 alpha-3-sulphate, with a concomitant reduced MCR of oestradiol-17 beta from plasma. 4. The metabolic defect was corrected by feeding the control diet. 5. Because the metabolic defect observed in calciferol deficiency occurred in Ca deficiency in a more severe form, we conclude that the more immediate cause was calcium rather than calciferol deficiency. To our knowledge, this is the first observation of a calcium-deficient effect on oestrogen sulphate metabolism in vivo.