Using a system that expresses a constitutively kinase-active c-Abl protein [c-Abl(KA)], we identified the protein IkappaBalpha as a novel substrate of c-Abl. This kinase-substrate relationship is not only confirmed at the level of endogenous proteins but is also supported by a physical interaction between the two proteins. Interestingly, the association of c-Abl with IkappaBalpha, which is detectable in the form of nonphosphorylated proteins, is remarkably enhanced by an inducible binding of tyrosine-phosphorylated IkappaBalpha to the c-Abl SH2 domain. In contrast to the serine 32/34 phosphorylation that triggers ubiquitination and degradation of IkappaBalpha, c-Abl-mediated phosphorylation at tyrosine 305 is associated with an increase of the IkappaBalpha protein stability. Significantly, this activity is not shared by the oncogenic Bcr-Abl, because it is unique to the nuclear c-Abl. We also demonstrate that c-Abl targets the nuclear subpopulation of IkappaBalpha for phosphorylation and induces it to accumulate in the nucleus. As a consequence, NF-kappaB transcription activity is abolished, leading to an increased cellular sensitivity to the induction of apoptosis. The functional importance of c-Abl-mediated IkappaBalpha phosphorylation is highlighted by a loss of response of the IkappaBalpha(Y305F) protein to c-Abl-mediated regulation. Using cells expressing the c-Abl(KA) protein under the control of an inducible promoter, we demonstrate inactivation of the NF-kappaB-dependent cell survival pathway as one of the mechanisms for c-Abl-mediated apoptosis.