The objective of this research is to elucidate the mechanisms by which the metabolism of 25-hydroxyvitamin D3 in the kidney is regulated. One product of this metabolism, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is of particular interest as it is the very potent biologically active form of the parent vitamin D3. In order to explore the regulation of the production of 1,25(OH)2D3, and the other metabolite of 25-OH-D3, 24,25-(OH)2D3, primary cultures of chick kidney cells have been utilized. This system has and continues to yield important insights into how 1,25(OH)2D3 reduces its own production and induces that of 24,25(OH)2D3, and how parathyroid hormone might be involved in this process. In order to continue to make progress in understanding the cellular and molecular details, however, it is necessary to examine a homogeneous cell population. It is therefore, proposed to develop clonal cell lines of chick kidney cells, yielding homogeneous populations, which maintain the appropriate differentiated characteristics. In both primary cultures and cell lines the mechanism of action of 1,25(OH)2D3 will be studied by determining its intracellular localization, the nature of any specific binding components, the determination of any new gene products that might be made in response to the steroid. The stimulatory effect of PTH on 1,25(OH)2D3 production will be investigated in terms of its dependence on protein synthesis, its stimulation of cyclic AMP dependent protein kinase activity and the identification of possible phosphorylated proteins. In addition, other hormones and regulatory factors, such as calcitonin, prolactin, insulin and extracellular calcium and phosphorus will be tested for effects on 25-OH-D3 metabolism in primary cultures and cell lines. The results of these studies will advance our understanding of the nature of the production of the important steroid hormone, 1,25(OH)2D3 and its regulation, so that derangements of the system might be better anticipated and dealt with.