IκB kinase γ (IKKγ) (also known as NEMO, Fip-3, and IKKAP-1) is the essential regulatory component of the IKK complex; it is required for NF-κB activation by various stimuli, including tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1), phorbol esters, lipopolysaccharides, and double-stranded RNA. IKKγ is encoded by an X-linked gene, deficiencies in which may result in two human genetic disorders, incontinentia pigmenti (IP) and hypohidrotic ectodermal dysplasia with severe immunodeficiency. Subsequent to the linkage of IKKγ deficiency to IP, we biochemically characterized the effects of a mutation occurring in an IP-affected family on IKK activity and NF-κB signaling. This particular mutation results in premature termination, such that the variant IKKγ protein lacks its putative C-terminal Zn finger and, due to decreased mRNA stability, is underexpressed. Correspondingly, IKK and NF-κB activation by TNF-α and, to a lesser extent, IL-1 are reduced. Mutagenesis of the C-terminal region of IKKγ was performed in an attempt to define the role of the putative Zn finger and other potential functional motifs in this region. The mutants were expressed in IKKγ-deficient murine embryonic fibroblasts (MEFs) at levels comparable to those of endogenous IKKγ in wild-type MEFs and were able to associate with IKKα and IKKβ. Substitution of two leucines within a C-terminal leucine zipper motif markedly reduced IKK activation by TNF-α and IL-1. Another point mutation resulting in a cysteine-to-serine substitution within the putative Zn finger motif affected IKK activation by TNF-α but not by IL-1. These results may explain why cells that express these or similar mutant alleles are sensitive to TNF-α-induced apoptosis despite being able to activate NF-κB in response to other stimuli.