The Nramp1 gene controls macrophage resistance or susceptibility to several intracellular microorganisms; however, there is conflicting evidence regarding its role during infection with virulent Mycobacterium tuberculosis. Nitric oxide (NO) is a potent antimycobacterial agent produced by macrophages, which is also regulated by Nramp1. The in vitro ability of B10R (resistant) and B10S (susceptible) murine macrophages to inhibit M. tuberculosis H37Rv and to produce NO in response to infection and interferon-gamma (IFN-gamma) was compared. Infected B10R macrophages inhibited [3H]uracil incorporation by M. tuberculosis and produced higher amounts of NO than did B10S macrophages. IFN-gamma increased the inhibitory activity of both cells. Inhibition of M. tuberculosis by IFN-gamma-activated B10R macrophages was reversed by N(G)-monomethyl-L-arginine (N(G)MMA). L-arginine restored NO production and increased the antimycobacterial activity by IFN-gamma-stimulated N(G)MMA-treated macrophages. The Bcg/Nramp1 gene may regulate macrophage resistance or susceptibility to virulent M. tuberculosis by a differential capability of these cells to produce NO.