Embryonic chick duodenum can be maintained in organ culture in contact with a serum-free medium with excellent preservation of mucosal structure. The presence of vitamin D3 in the culture medium induces de novo synthesis of a calcium-binding protein (CaBP) and stimulates uptake and mucosal to serosal transport of calcium in the cultured intestine. Using this system, it was found that vitamin D3 in the culture medium elevated duodenal cyclic AMP concentration after only 3hr of incubation. This response was prior to induction of CaBP and stimulation of calcium accumulation. Following a decline to control levels at 6 hr, there was a continuous rise in duodenal cyclic AMP, paralleling CaBP synthesis and enhanced calcium uptake, throughout the 48-hr culture period. Addition of either dibutyryl cyclic AMP (dbcAMP) or theophylline to the medium at lmM stimulated 45Ca accumulation by the intestine regardless of the presence of vitamin D3,although stimulation was greatest in the presence of the sterol, and the effect of these agents was additive. While stimulating radiocalcium accumulation, both agents reduced the production of CaBP, a protein known to be involved in intestinal calcium transport. At 1 mM.Na butyrate, cyclic AMP or 5'-AMP was ineffective. It is unlikely that vitamin (D)3 itself had an action on membrane permeability independent of CaBP induction since actinomycin D completely blocked both CaBP synthesis and stimulation of calcium translocation. The action of dbcAMP on calcium uptake was observable within 3 hr and persisted at approximately the same intensity throughout the entire culture period (48 hr). The CaBP inhibiting action of dbcAMP began only after 24 hr. Both total protein and DNA content of the cultured tissue increased in the presence of dbcAMP. dbcAMP stimulated both uptake and mucosal to serosal transport of calcium in duodena incubated in the presence or absence of vitamin D3 and dbcAMP effects were additive to some extent. The results indicate an interaction involving vitamin D3, CaBP, adenylate cyclase, and the intestinal calcium absorptive mechanism. (Endocrinology94: 1607, 1974)