We have shown that several lipids can modulate the macrophage innate immune response against mycobacteria and enhance their killing. Since NF-kappaB is required for mycobacterial killing, we tested the ability of lipids to activate NF-kappaB in uninfected macrophages and those infected with mycobacteria. In uninfected cells, sphingomyelin (SM), phosphatidylinositol-4-phosphate (PIP) and arachidonic acid (AA) enhanced NF-kappaB activation and the cell surface expression of CD69, a macrophage activation marker regulated by NF-kappaB. Sphingosine (Sph), sphingosine-1-phosphate (S1P), diacylglycerol (DAG), eicosapentanoic acid (EPA) and phosphatidyl choline (PC) failed to activate either NF-kappaB or CD69. Ceramide (Cer) activated CD69 expression without activating NF-kappaB. In Mycobacterium smegmatis-infected cells, NF-kappaB was transiently activated in a manner that was enhanced by SM, PIP and AA. In contrast Mycobacterium avium mostly repressed NF-kappaB activation and only SM and AA could induce its partial activation. While lipids that activate NF-kappaB in uninfected cells tend to kill mycobacteria in macrophages Sph and S1P failed to activate NF-kappaB under most conditions but nevertheless enhanced killing of M. smegmatis, M. avium and M. tuberculosis H37Rv. Our results argue that both NF-kappaB-dependent and -independent mechanisms are involved in macrophage killing of mycobacteria and that both mechanisms can be enhanced by selected lipids.