The time-course of the changes in blood ionized calcium, and in plasma 1,25 dihydroxycholecalciferol (1,25(OH)2D3) concentrations and its free index were studied in hens following suppression and resumption of shell formation and throughout the laying cycle in hens laying hard-shelled eggs, in hens fed a low or normal calcium diet and in hens laying shell-less eggs. The respective roles of the calcium needs for shell formation and of the reproductive status in regulation of 1,25(OH)2D3 production were analysed. Plasma 1,25(OH)2D3 decreased 3 hr after suppression of shell formation following premature egg expulsion and remained lower than that of hens laying hard-shelled eggs when premature expulsion of the eggs was continued for several days. Circulating 1,25(OH)2D3 tended to increase progressively when shell formation was resumed. Ablation of the parathyroid glands abolished this increase. In hens laying hard-shelled eggs, the plasma 1,25(OH)2D3 was higher during the period of shell secretion. Feeding hens a low calcium diet (1.2%) caused a marked increase in the plasma 1,25(OH)2D3. Ionized calcium levels tended to show reciprocal changes to plasma 1,25(OH)2D3 decreasing when calcification took place and increasing after its suppression. In hypercalcemic hens laying shell-less eggs and fed a 3.5% Ca diet, the plasma 1,25(OH)2D3 was at a high level 4 hr after ovulation and diminished thereafter. This additive stimulation does not, therefore, involve the parathyroid gland and may involve hormonal changes induced by ovulation. Vitamin D binding protein (DBP) in the plasma was at a high level in mature hens and was not affected by shell formation. Consequently, the free 1,25(OH)2D3 index fluctuated in parallel with total level of this hormone in mature hens. It is concluded that the calcium demand for shell formation modulates, in the short term, plasma 1,25(OH)2D3, via the homeostatic regulation of blood calcium by PTH, but that a large part of its increase is independent of PTH and is associated with the endocrine events concomitant with ovulation.