It has been demonstrated from studies using NF-kappa B inhibitors that NF-kappa B may be involved in the iNOS induction stimulated by cytokines and/or lipopolysaccharide (LPS) in various cell types and tissues. However, the actions of the inhibitors are less selective and highly cytotoxic, We constructed stable clones of C6 cells transfected with two types of I kappa B alpha mutant genes (I kappa B alpha(SS-->AA); Ser-32/36 to Ala-32/36, I kappa B alpha(KK-->RR);Lys-21/22 to Arg-21/22). I kappa B alpha(SS-->AA) strongly inhibited (1) LPS-, IL-1 beta-, and TNF-alpha-induced nuclear translocation and DNA binding of NF-kappa B to the kappa B site; and (2) iNOS induction stimulated by LPS or IL-1 beta plus IFN-gamma, These results indicate that NF-kappa B plays a critical role in cytokines and/or LPS-induced iNOS induction. Surprisingly, similar to the endogenous I kappa B alpha, I kappa B alpha(KK-->RR) was degraded by various stimuli, and proteasome inhibitors blocked this event. These results suggest that another Lys residue(s), other than Lys-21/22, may be required for the ligand-induced I kappa B alpha degradation by the ubiquitin-proteasome pathway.