Vitamin D3-3H metabolism was studied in 4 normal adult subjects before and during periods of prednisone administration. The biological activity of plasma vitamin D3 and its metabolites was assayed by in vitro measurements of 45Ca accumulation by duodenal slices. In each instance prednisone induced a decrease in the plasma half-time of vitamin D3-3H, a decrease in the accumulation of a biologically active vitamin D3 metabolite and the rapid appearance of chloroform-soluble biologically inactive metabolites without concomitant alterations in water-soluble vitamin D3 metabolites. These experiments support the conclusion that the observed antagonism of glucocorticoids to the action of vitamin D in man is related to the rapid turnover of vitamin D3, a diminished production of a biologically active vitamin D metabolite and the subsequent decrease in the intestinal absorption of calcium.