Hormonal vitamin D3 is a major regulator of calcium metabolism and is involved in basic cellular processes, such as those of proliferation and differentiation. These actions are mediated via an intracellular vitamin D3 receptor (VDR), which is a member of the evergrowing steroid hormone receptor superfamily. The interaction between the vitamin D3 ligand and its receptor is thought to be through a classic steroid hormone mechanism. It is notable, however, that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] has also been documented as an agent that directly up-regulates endogenous VDR in both intact animals and cultured cells. In this report, we confirm that the levels of recombinantly expressed VDR produced in transiently transfected COS-1 cells also increase several-fold when the cells are treated with 1,25-(OH)2D3. Additionally, we show that a similar pattern is exhibited in a Saccharomyces cerevisiae expression system. This indicates that the mechanism for VDR up-regulation is conserved in both yeast and mammalian cells. Our results show that up-regulation by 1,25-(OH)2D3 is specific to the VDR, and that 1,25-(OH)2D3 does not affect other expressed receptor proteins, such as those for estrogen and progesterone. Finally, we demonstrate that the mechanism of up-regulation apparently occurs at the level of the protein and is most likely due to altered stability of the occupied receptor. Our observations lead us to propose that in addition to the classically viewed role of hormone in receptor activation, 1,25-(OH)2D3 may serve to amplify signal response via homologous up-regulation.