Constitutive nuclear factor kappaB (NF-κB) activity protects quiescent mature immune cells from spontaneous apoptosis. Here, we examined whether NF-κB exerts its antiapoptotic function in these cells through the control of Bcl-2 family proteins. Specific pharmacologic inhibitors of NF-κB were used to achieve total NF-κB inactivation in quiescent human blood lymphocytes, granulocytes, and monocytes. NF-κB inhibition induced drastic lymphocyte and granulocyte apoptosis, but only moderate monocyte apoptosis. T- and B-cell apoptosis was slow and associated with a gradual down-regulation of the prosurvival Bcl-2 family proteins Bcl-x_L and Bcl-2, respectively. By contrast, granulocyte apoptosis was fast and accompanied by a rapid cellular accumulation of Bcl-x_S, the proapoptotic Bcl-x isoform that is generated from alternative splicing of the bcl-x pre-mRNA. Finally, antisense bcl-x_L and bcl-2knockdown in T and B cells, respectively, and induction of Bcl-x_S expression in granulocytes through antisense oligonucleotide-mediated redirection of bcl-x pre-mRNA splicing were sufficient to induce significant apoptosis in these cells. Taken together, these results reveal that basal NF-κB activity preserves homeostasis of quiescent mature lymphocytes and granulocytes through regulation of distinct members of the Bcl-2 family. This study sheds light on the constitutive mechanisms by which NF-κB maintains defense integrity.