Historically, administration of vitamin D has been considered beneficial in the treatment of tuberculosis. The interaction of this vitamin [i.e., 1,25-dihdroxyvitamin D(3) [1,25(OH)(2)D(3)]] with the antitubercular immune response, however, is not clear. In the present study, in vitro recall responses of peripheral blood mononuclear cells (PBMC) from cattle infected with Mycobacterium bovis were used to study the immune-modulatory effects of 1,25(OH)(2)D(3) on M. bovis-specific responses in vitro. Addition of 1 or 10 nM 1,25(OH)(2)D(3) inhibited M. bovis-specific proliferative responses of PBMC from M. bovis-infected cattle, affecting predominantly the CD4(+) cell subset. In addition, 1,25(OH)(2)D(3) inhibited M. bovis-specific gamma interferon (IFN-gamma) production yet enhanced M. bovis-specific nitric oxide (NO) production. Lymphocyte apoptosis, measured by flow cytometry using annexin-V staining, was diminished by addition of 1,25(OH)(2)D(3) to PBMC cultures. These findings support the current hypothesis that 1,25(OH)(2)D(3) enhances mycobacterial killing by increasing NO production, a potent antimicrobial mechanism of activated macrophages, and suggest that 1,25(OH)(2)D(3) limits host damage by decreasing M. bovis-induced IFN-gamma production.