Nitric oxide (NO(.)) produced by inducible nitric oxide synthase (iNOS) is an important host defense molecule against Mycobacterium tuberculosis in mononuclear phagocytes. The objective of this study was to determine the role of the IkappaBalpha kinase-nuclear factor kappaB (IKK-NF-kappaB) signaling pathway in the induction of iNOS and NO(.) by a mycobacterial cell wall lipoglycan known as mannose-capped lipoarabinomannan (ManLAM) in mouse macrophages costimulated with gamma interferon (IFN-gamma). NF-kappaB was activated by ManLAM as shown by electrophoretic mobility shift assay, by immunofluorescence of translocated NF-kappaB in intact cells, and by a reporter gene driven by four NF-kappaB-binding elements. Transduction of an IkappaBalpha mutant (Ser32/36Ala) significantly inhibited NO(.) expression induced by IFN-gamma plus ManLAM. An activated SCF complex, a heterotetramer (Skp1, Cul-1, beta-TrCP [F-box protein], and ROC1) involved with ubiquitination, is also required for iNOS-NO(.) induction. Two NF-kappaB-binding sites (kappaBI and kappaBII) present on the 5'-flanking region of the iNOS promoter bound ManLAM-induced NF-kappaB similarly. By use of reporter constructs in which one or both sites are mutated, both NF-kappaB-binding positions were essential in iNOS induction by IFN-gamma plus ManLAM. IFN-gamma-induced activation of the IRF-1 transcriptional complex is a necessary component in host defense against tuberculosis. Although the 5'-flanking region of the IRF-1 promoter contains an NF-kappaB-binding site and ManLAM-induced NF-kappaB also binds to this site, ManLAM was unable to induce IRF-1 expression. The influence of mitogen-activated protein kinases on IFN-gamma plus ManLAM induction of iNOS-NO(.) is not due to any effects on ManLAM induction of NF-kappaB.