CYP24A1 functions in vitamin D target tissues to degrade 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). Thus, the concentration of this enzyme and the regulation of its expression is a primary determinant of the overall biological activity of 1,25(OH)(2)D(3) within cells. The principle regulator of CYP24A1 expression is 1,25(OH)(2)D(3) itself, which functions through the vitamin D receptor to upregulate the transcriptional activity of the Cyp24a1 gene. In this report, we explore the mechanism of this regulation using recently developed ChIP-chip and ChIP-seq techniques that permit an unbiased search for enhancer elements that participate in this transcriptional control. Our studies both confirm a regulatory region defined earlier and located proximal to the transcriptional start site (TSS) of mouse Cyp24a1 (-160 and -265nt) and identify a novel intergenic region located downstream of the transcription unit that contains two enhancers (+35 and +37kb) that facilitate 1,25(OH)(2)D(3)-dependent upregulation of Cyp24a1 expression. Interestingly, while C/EBPβ also binds under basal conditions to a site located immediately upstream of the Cyp24a1 promoter (-345nt), occupancy by this factor is strikingly increased following 1,25(OH)(2)D(3) treatment. The locations and activities of these regulatory regions that mediate 1,25(OH)(2)D(3) actions were confirmed in mice in vivo. We conclude that the mechanism through which 1,25(OH)(2)D(3) induces the CYP24A1 enzyme, thereby autoregulating its own destruction, involves both promoter-proximal as well as downstream-distal enhancers. These findings highlight new concepts regarding the molecular mechanism of action of 1,25(OH)(2)D(3) and other hormonal regulators.