Although the essential role of tumor necrosis factor (TNF) in the control of intracellular bac-terial infection is well established, it is uncertain whether the related cytokines lymphotoxin-α (LTα₃) and lymphotoxin-β (LTβ) have independent roles in this process. Using C57Bl/6 mice in which the genes for these cytokines have been disrupted, we have examined the relative contribution of secreted LTα₃ and membrane-bound LTβ in the host response to aerosol Mycobacterium tuberculosis infection. To overcome the lack of peripheral lymph nodes in LTα^−/− and LTβ^−/− mice, bone marrow chimeric mice were constructed. LTα^−/− chimeras, which lack both secreted LTα₃ and membrane-bound LTβ (LTα1β2 and LTα2β1), were highly susceptible and succumbed 5 wk after infection. LTβ^−/− chimeras, which lack only the membrane-bound LTβ, controlled the infection in a comparable manner to wild-type (WT) chimeric mice. T cell responses to mycobacterial antigens and macrophage responses in LTα^−/− chimeras were equivalent to those of WT chimeras, but in LTα^−/− chimeras, granuloma formation was abnormal. LTα^−/− chimeras recruited normal numbers of T cells into their lungs, but the lymphocytes were restricted to perivascular and peribronchial areas and were not colocated with macrophages in granulomas. Therefore, LTα₃ is essential for the control of pulmonary tuberculosis, and its critical role lies not in the activation of T cells and macrophages per se but in the local organization of the granulomatous response.