5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of proinflammatory leukotrienes. The 5-LO gene is a primary target of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) and its expression is prominently increased during myeloid cell differentiation. Since no functional vitamin D response element (VDRE) has been reported for this gene so far, we performed in silico screening of the whole 5-LO gene area (84 kb, including 10 kb promoter region) and identified 22 putative VDREs. Both gelshift and reporter gene assays identified four of these candidates as functional VDREs. Their approximate positions are -2,250 (promoter), +21,400 (intron 2), +42,000 (intron 4) and +50,600 (intron 5) in relation to the transcription start site (TSS). Remarkably, the VDRE at position +42,000 is one of the strongest known VDREs of the human genome. Chromatin immunoprecipitation (ChIP) assays demonstrated simultaneous association of vitamin D receptor (VDR), retinoid X receptor (RXR) and RNA polymerase II (Pol II) to the 5-LO gene regions containing two of these four putative VDREs. This indicates DNA looping of the TSS to even very distant gene regions. In summary, we suggest that the upregulation of the primary 1alpha,25(OH)(2)D(3) target 5-LO is mediated in vivo by a prominent VDRE in intron 4.