Stimulation of cells with tumor necrosis factor alpha (TNFalpha) triggers NF-kappaB1 p105 proteolysis, releasing associated Rel subunits to translocate into the nucleus and modulate target gene expression. Phosphorylation of serine 927 within the p105 PEST region by the IkappaB kinase (IKK) complex is required to promote p105 proteolysis in response to TNFalpha stimulation. In this study, the role of the p105 death domain (DD) in signal-induced p105 proteolysis is investigated. Endogenous p105 is shown to interact with the IKK complex in HeLa cells, and transient transfection experiments in 293 cells indicate that each of the catalytic components of the IKK complex, IKK1 and IKK2, can bind to p105. Interaction of p105 with both IKK1 and IKK2 is substantially reduced by deletion of the p105 DD or introduction of a specific point mutation (L841A) into the p105 DD homologous to the lpr mutation in Fas. Phosphorylation of immunoprecipitated p105 on serine 927 by purified recombinant IKK1 or IKK2 protein in vitro is dramatically reduced in both DD mutants relative to wild type. Furthermore, both of the DD mutations significantly impair the ability of low concentrations of IKK2 to induce p105 serine 927 phosphorylation and proteolysis in transiently transfected 3T3 cells. However, high levels of transiently expressed IKK2 bypass the requirement for the p105 DD to induce p105 serine 927 phosphorylation. Finally, p105 serine 927 phosphorylation by the endogenous IKK complex after TNFalpha stimulation and subsequent p105 proteolysis is blocked in both p105 DD mutants when stably expressed in HeLa cells. Thus, the p105 DD acts as a docking site for IKK, increasing its local concentration in the vicinity of the p105 PEST region and facilitating efficient serine 927 phosphorylation.