- Background. In a previous study we demonstrated the inhibitory effect of 1,25-dihydroxyvitamin D (1,25(OH) 2 D 3 ) and its less calcaemic analog 1,24(OH) 2 D 2 on the production of tumour necrosis factor alpha (TNFa) by human peritoneal macrophages. The aim of the present study is to examine whether this vitamin D inhibition of TNFa is mediated by its major transcription factor, nuclear factor-κB (NFKB). Methods. Murine macrophage cells (P388D1) were incubated with 10 -7 M 1,25(OH) 2 D 3 or 1,24(OH) 2 D 2 and then stimulated with lipopolysaccharide. NFKB activity was assayed using a reporter gene and by electrophoretic mobility shift assay (EMSA). In addition, we evaluated mRNA and protein levels of NFκB-p65 and of IicBa, a potent NFκB inhibitor, and phosphorylated IκBα. Results. Both 1,25(OH) 2 D 3 and 1,24(OH) 2 D 2 induced a 60% reduction of TNFa secretion. By using a reporter gene and EMSA we found that vitamin D markedly reduced NFKB activity. 1,25(OH) 2 D 3 or 1,24(OH) 2 D 2 decreased NFκB-p65 levels in the nucleus and increased NFκB-p65 levels in the cytosol; no changes were observed in the total levels of NFκB-p65 protein and mRNA. Concurrently, vitamin D induced a significant increase in mRNA and protein levels of IκBα (∼6.5- and 4.5-fold, respectively). Elevated levels of IκBα can be explained by the vitamin D-induced prolongation of IκBα-mRNA half-life from 110 to 190min and by the decrease in IicBa phosphorylation. Conclusions. Vitamin D up-regulates IicBa levels by increasing mRNA stability and decreasing IicBa phosphorylation. The increase in IκBα levels reduces nuclear translocation of NFKB and thereby downgrades its activity. Since NFKB is a major transcription factor of inflammatory mediators, these findings suggest that the less-calcaemic analog, 1,24(OH) 2 D 2 may be effective as an anti-inflammatory therapeutic agent.