The active vitamin D metabolite 1α,25-dihydroxyvitamin D (1,25[OH]2 D) potently inhibits dendritic cell (DC) priming of T-cell activation, suggesting that it mediates a homeostatic role in this context. Therefore, careful regulation of 1,25[OH]2 D levels is necessary to avoid inappropriate inhibition of T-cell activation. Cell-autonomous control of vitamin D activity can be modulated by the action of the vitamin D-activating and -inactivating hydroxylases, CYP27B1 and CYP24A1, respectively. We show that in comparison to macrophages, human monocyte-derived DCs exhibit significantly less activation of 25[OH]D to 1,25[OH]2 D, and that DCs predominantly express a truncated CYP27B1 transcript that may contribute to the deficiency in activation of vitamin D. Furthermore, in response to stimulation with 1,25[OH]2 D, up-regulation of the inactivating enzyme CYP24A1 curtailed the functional effects of vitamin D in DCs, but not macrophages. Production of 1,25[OH]2 D by macrophages was adequate to induce expression of vitamin D-responsive genes by DCs, inhibit DC maturation in response to innate immune stimulation and DC-dependent T-cell responses. Our data suggest that in comparison to macrophages, differential regulation of hydroxylases limits autocrine vitamin D activity in DCs, and that paracrine activation of vitamin D exerts a more potent mechanism for homeostatic control of DC function. This article is protected by copyright. All rights reserved.