A number of different kinases have been implicated in NF-kappa B regulation and survival function. Here we investigated the molecular cross-talk between glycogen synthase kinase-3 beta (GSK-3 beta) and the p105 precursor of the NF-kappa B p50 subunit. GSK-3 beta forms an in vivo complex with and specifically phosphorylates NF-kappa B1/p105 at Ser-903 and Ser-907 in vitro. In addition, the p105 phosphorylation level is reduced in fibroblasts lacking GSK-3 beta as compared with wild-type cells. GSK-3 beta has a dual effect on p105: it stabilizes p105 under resting conditions and primes p105 for degradation upon tumor necrosis factor (TNF)-alpha treatment. Indeed, constitutive processing of p105 to p50 occurs at a higher rate in cells lacking GSK-3 beta with respect to wild-type cells and can be reduced upon reintroduction of GSK-3 beta by transfection. Moreover, p105 degradation in response to TNF-alpha is prevented in GSK-3 beta-/- fibroblasts and by a Ser to Ala point mutation on p105 at positions 903 or 907. Interestingly, the increased sensitiveness to TNF-alpha-induced death occurring in GSK-3 beta-/- fibroblasts, which is coupled to a perturbation of p50/105 ratio, can be reproduced by p105 silencing in wild-type fibroblasts.