Regulation of interleukin-8 (IL-8) gene transcription occurs mainly through the sequences -94 to -71 of the 5'-flanking region of the IL-8 gene, involving the transcription factors nuclear factor for interleukin-6 (NF-IL-6) and nuclear factor kappaB (NF-kappaB). The human melanoma cell line A3 was derived from G-361 cells by stable transfection with an IL-8 promoter-luciferase construct containing these sequences. 1alpha,25-Dihydroxyvitamin D3 (calcitriol) repressed IL-8 promoter activity induced by tumor necrosis factor-alpha (TNF-alpha) by 50%, compared to 30% inhibition using dexamethasone, an effect consistent with its effect on TNF-alpha-induced IL-8 release and IL-8 mRNA levels. A variety of vitamin D metabolites caused the same repressive effect on IL-8 promoter activation as calcitriol. However, only those metabolites which were able to transactivate a classical vitamin D response element had the ability to repress IL-8 promoter activation, suggesting that this repression is mediated via vitamin D receptor (VDR). Furthermore, overexpression of VDR in the parental G-361 cell line enhanced the repressive effect of calcitriol on activation of the IL-8 promoter by either TNF-alpha stimulation or overexpression of the NF-kappaB subunit p65. Electrophoretic mobility shift assays using nuclear extracts from A3 cells showed that calcitriol decreased the abundance of nuclear factors bound to the NF-kappaB binding site of the IL-8 promoter and this reduced binding of NF-kappaB proteins presumably contributes to its inhibitory action.