The C-14 epimers of vitamin D, 14-epi-25-hydroxyvitamin D3 (4) and 14-epi-1 alpha, 25-dihydroxyvitamin D3 (5), were synthesized, and their isomerization via [1,7]-sigmatropic hydrogen shifts to the corresponding previtamin forms (4' and 5', respectively) was studied. The activation parameters of the [1,7]-sigmatropic hydrogen shifts were found to be similar to those of other vitamin D analogues, although epimerization at C-14 shifts the equilibrium of the triene chromophore to the previtamin form. The in vivo biological activities of 4, 4', 5, and 5' in the chick in terms of their ability to elicit intestinal calcium absorption and bone calcium mobilization were determined. These vitamin D analogues, the first in the natural steroid series modified at the C-14 position, were essentially devoid of activity. The relative competitive indices (RCIs), derived in an in vitro assay reflecting the ability of these analogues to bind to the chick intestinal nuclear receptor, were determined. Analogues 4, 4', 5, and 5' had RCI values of 0.08, 0.01, 15, and 1.6, respectively, in comparison to the natural ligand, 1 alpha, 25-dihydroxyvitamin D3 (3), whose value is 100 by definition. Thus, the in vivo and in vitro data were somewhat at variance, particularly for 5, which bound significantly to the chick intestinal receptor. In vitro binding studies with the human serum vitamin D binding protein (DBP) were also conducted. The RCI values for human DBP reflects the ability of an analogue to bind to this protein in comparison to the hormone 3, whose value is 100. The measured RCI values for 4, 4', 5, and 5' were 3450, 90, 12, and 2.2, respectively. It is noteworthy that analogue 4 binds approximately 35 times more effectively than the parent hormone 3, but approximately 20 times less effectively than 25-hydroxyvitamin D3 (2).