Vitamin D deficiency is associated with an increased risk for tuberculosis infection. Studies using in vitro systems indicate that 1,25-dihydroxyvitamin D(3) [i.e. 1,25(OH)(2)D(3)], the most active form of the vitamin, enhances mycobacterial killing by increasing nitric oxide (NO) production. To evaluate concurrently the role of 1,25(OH)(2)D(3) and NO on the host response to tuberculosis infection, mice deficient in NO synthase 2 (NOS2(-/-)) and/or vitamin D were aerosol-challenged with Mycobacterium bovis and subsequently evaluated for mycobacterial colonization and lesion formation. Infected NOS2(-/-) mice developed severe necrotizing pyogranulomatous inflammation of the lungs with heavy M. bovis colonization and systemic dissemination of the bacillus. Colonization and lung lesion area of NOS2(-/-) mice exceeded that of NOS2(+/+) mice. Additionally, disease progression was more rapid in NOS2(-/-) mice than in NOS2(+/+) mice. Lung colonization and lesion area of vitamin D deficient mice exceeded that of vitamin D replete mice, regardless of NOS2 phenotype. However, effects of vitamin D on colonization, but not lesion area, were more pronounced in NOS2(+/+) mice than in NOS2(-/-) mice. These findings are consistent with the current hypothesis that 1,25(OH)(2)D(3) enhances mycobacterial killing through a NO-dependent mechanism. As responses of NOS2(-/-) mice were affected by 1,25(OH)(2)D(3) deficiency, albeit to a lesser extent than were those of NOS2(+/+) mice, NO-independent actions of 1,25(OH)(2)D(3) also likely exist.