1 alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], the biologically active metabolite of vitamin D3, inhibited synthesis of gamma-interferon (IFN-gamma) by phytohemagglutinin-activated peripheral blood lymphocytes (PBLs). A significant reduction of IFN-gamma protein levels in PBL culture medium was achieved with a physiologic 1,25-(OH)2D3 concentration (0.1 nM). 1,25-(OH)2D3 also inhibited accumulation of IFN-gamma mRNA in activated PBLs in a dose-dependent fashion. The ability of 1,25-(OH)2D3 to modulate IFN-gamma protein synthesis was unaltered in the presence of high concentrations of recombinant human interleukin 2. The suppression of IFN-gamma synthesis by PBLs was specific for 1,25-(OH)2D3; the potencies of other vitamin D3 metabolites were correlated with their affinities for the cellular 1,25-(OH)2D3 receptor. The time course of 1,25-(OH)2D3 receptor expression in phytohemagglutinin-activated PBLs was correlated with the time course of 1,25-(OH)2D3-mediated inhibition of IFN-gamma synthesis. In selected experiments, T-lymphocyte-enriched cell preparations were utilized. In these experiments, 1,25-(OH)2D3 was equally active as in PBL preparations. Finally, we examined the effects of 1,25-(OH)2D3 on the constitutive IFN-gamma production by two human T-lymphocyte lines transformed by human T-lymphotropic virus type I. The cell lines were established from a normal donor (cell line S-LB1) and from a patient with vitamin D-dependent rickets type 2 (cell line Ab-VDR). IFN-gamma synthesis by S-LB1 cells was inhibited in a dose-dependent fashion by 1,25-(OH)2D3, whereas IFN-gamma synthesis by Ab-VDR cells was not altered by 1,25-(OH)2D3. The data presented in this study provide further evidence for a role of 1,25-(OH)2D3 in immunoregulation.