Evidence is presented that in vitamin D deficiency there are aberrations of estrogen metabolism in the laying hen. Five White Leghorn hens, 35 weeks of age, all laying soft shelled eggs as a result of complete withdrawal of supplemental vitamin D3 (D3) from the diet, were found to have a 3-fold higher molar ratio of plasma estradiol-17 beta-3-sulfate (E2 beta-3S) to estradiol-17 beta (E2 beta) compared to five control laying hens fed a D3-supplemented diet. A time course tracer study with [3H]E2 beta administered iv revealed an abnormally high accumulation of [3H]E2 beta-3S in plasma, and concomitantly, a markedly reduced formation of [3H]estradiol-17 alpha-3-sulfate [( 3H]E2 alpha-3S) compared with the control hens. This observation suggests a defect in the conversion of [3H]E2 beta-3S to [3H]E1S, previously shown to be a major pathway for E2 beta metabolism (E2 beta E2 beta-3S in equilibrium E1S E2 alpha-3S). As a result, the MCR of [3H]E2 beta from plasma of the D3-deficient hens was about 15% below normal. The calculated production rate of E2 beta was reduced even more, by 60%. However, the reduced production rate could have been related to the lengthening of the ovulatory cycle due to vitamin D deficiency. It is concluded that vitamin D deficiency affects the biosynthesis and catabolism of E2 beta in the laying hen.