In a previous study we demonstrated a down-regulatory effect of vitamin D active metabolite (1,25(OH)(2)D(3)) and its vitamin D(2) analog (1,24(OH)(2)D(2)) on TNFalpha expression in macrophages. We also found an inhibitory effect in the physiological concentration (10(-10)M) of 1,25(OH)(2)D(3) which was dose-dependent. This down-regulation, caused by the decrease in NFkappaB activity by 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2), was demonstrated in P388D1 cells transfected with NFkappaB reporter gene (p NFkappaB-Luc) and by EMSA. In our present study we investigated the processes leading to reduced NFkappaB activity on P388D1 cells. A decrease in nuclei NFkappaB-p65 and an increase in cytosolic NFkappaB-p65, were measured, while no changes in total NFkappaB-p65 mRNA and protein levels were observed. Simultaneously, a significant increase in both mRNA and protein levels of the NFkappaB-cytosolic inhibitor, IkappaBalpha, were determined. The half-life of IkappaBalpha-mRNA increased, with a parallel decrease in the phosphorylation of its protein, as the first step of ubiquitinization and degradation. The present results demonstrate that 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2) inhibit TNFalpha expression in macrophages, by increasing IkappaBalpha and decreasing NFkappaB activity. Since NFkappaB is a major transcription factor for TNFalpha and other inflammatory mediators, these findings suggest that 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2) may be used therapeutically as anti-inflammatory agents.