Maintenance of embryonic chick duodenum in organ culture in serum-free medium, responsive to vitamin D (or 1 alpha, 25-(OH)2-D3) and many analogues, with excellent preservation of mucosal structure, has been achieved. It is proposed to further investigate the regulation of synthesis and physiological role(s) of the vitamin D3-induced calcium-binding protein (CaBP) in the calcium adsorptive mechanism under the precisely controllable in vitro conditions, completely isolated from systemic influence, made possible only through the use of several variations of this unique system. Experiments to date with this technique have established that vitamin D (or 1 alpha, 25-(OH)2-D3), and numerous analogues, induce de novo synthesis of CaBP and stimulate transmucosal calcium transport by a calcium -dependent mechanism involving the adenyl cyclase system. Reconstitution experiments with purified CaBP have proven the involvement of this protein in the calcium transport process. Considerable further investigation of the interaction between vitamin D, CaBP, calcium and cyclic AMP will be undertaken. In addition, the relevance of a recently observed 1 alpha, 25-(OH)2-D3-induced, cAMP-dependent phosphoprotein kinase in the cultured duodenum will be vigorously pursued. The effects of a variety of agents, known to influence calcium adsorption in vivo, will be investigated. Among these are cortisol, recently shown to interact with vitamin D in CaBP biosynthesis and function in the system, diphenylhydantoin, shown to directly inhibit the intestinal calcium adsorptive mechanism, and other physiological and pharmacological agents, including several carcinogens, one of which was recently shown to interfere with CaBP biosynthesis. Attempts will be made to isolate, and use in reconstitution experiments, CaBP-mRNA. Finally, work with synthetic 1-alpha, 25-(OH)x-D3 indicates that this system could be used as a highly sensitive, specific, and relatively simple bioassay for this biologically significant steroid.