The side chain of vitamin D3 is hydroxylated in a sequential manner by cytochrome P450scc (CYP11A1) to form 20-hydroxycholecalciferol, which can induce growth arrest and differentiation of both primary and immortalized epidermalkeratinocytes. Since nuclear factor-kB (NF-kB) plays a pivotal role in the regulation of cell proliferation, differentiation andapoptosis, we examined the capability of 20-hydroxycholecalciferol to modulate the activity of NF-kB, using 1,25-dihydroxycholecalciferol (calcitriol) as a positive control. 20-hydroxycholecalciferol inhibits the activation of NFkB DNAbinding activity as well as NF-kB-driven reporter gene activity in keratinocytes. Also, 20-hydroxycholecalciferol inducedsignificant increases in the mRNA and protein levels of the NF-kB inhibitor protein, IkBa, in a time dependent manner, whileno changes in total NF-kB-p65 mRNA or protein levels were observed. Another measure of NF-kB activity, p65 translocationfrom the cytoplasm into the nucleus was also inhibited in extracts of 20-hydroxycholecalciferol treated keratinocytes.Increased IkBa was concomitantly observed in cytosolic extracts of 20-hydroxycholecalciferol treated keratinocytes, asdetermined by immunoblotting and immunofluorescent staining. In keratinocytes lacking vitamin D receptor (VDR), 20-hydroxycholecalciferol did not affect IkBa mRNA levels, indicating that it requires VDR for its action on NF-kB activity.Comparison of the effects of calcitrol, hormonally active form of vitamin D3, with 20-hydrocholecalciferol show that bothagents have a similar potency in inhibiting NF-kB. Since NF-kB is a major transcription factor for the induction ofinflammatory mediators, our findings indicate that 20-hydroxycholecalciferol may be an effective therapeutic agent forinflammatory and hyperproliferative skin diseases.