The calcium-regulating hormone 1,25-dihydroxyvitamin D3[1,25(OH)2D3] is recognized as an immunomodulator affecting the activities of macrophages and lymphocytes. We have shown that macrophages harvested from vitamin D-deficient mice (-D MPs) exhibit impaired phagocytic and tumoricidal activities as compared with control cells ( + D MPs), and that bone marrow-derived macrophage (BMDM) differentiation is modulated by 1 , 2 5 ( 0 H ) ~D ~. The release of tumor necrosis factor-a (TNF-a) by macrophages is considered a major mechanism by which these cells exert their tumoricidal function. This cytokine was also implicated in modulation of bone resorption. In the present study we examine the role of 1 ,25(OH)2D3 in TNF-a synthesis and release. BMDMs were harvested from + D and -D mice, cultured in vitro, and their conditioned media were analyzed for the presence of TNF-a. BMDMs did not release measurable amounts of TNF-a without stimulation. Addition of endotoxin (LPS) to the cultures resulted in a marked stimulation of TNF-a release. 1,25(OH)2D3 increased the stimulatory action of LPS, but failed to elicit a stimulatory effect in the absence of LPS. The use of another macrophage activator, interferon-? (IFN-y), yielded essentially similar results. + D and -D mice were injected with LPS and TNF-a levels in the serum were measured. A marked reduction (-fourfold) in the TNF-a levels was observed in the serum of -D mice as compared with + D mice. Western blot and immunoprecipitation analyses suggested that the main effect of 1 ,25(OH)2D3 is on TNF-a synthesis. O u r findings suggest that 1 ,25(OH)2D3 plays a role in the regulation of TNF-a secretion by mononuclear phagocytes. I 1994 Wiley-Liss, Inc