Introduction: Histone deacetylase inhibitors (HDI) fail to induce apoptosis in NSCLC relating, in part, to the activation of the anti-apoptotic transcription factor NF-kB in a PI3K/Akt dependent manner. We hypothesize that 1) the HDI, SAHA, activates NF-kB through p65 acetylation and that 2) NF-kB inhibition will sensitize NSCLC to SAHA-induced apoptosis. Methods: Four tumorigenic NSCLC cell lines were treated with SAHA, the PI3K/Akt inhibitor LY294002 or both drugs. NF-kB activity was determined by luciferase assays and by RT-PCR of the NF-kB-dependent genes Il-8, cIAP2, Bcl-XL, and Bfl/A1. Luciferase assays were repeated in cells overexpressing either the active NF-kB subunit p65wt or a p65K310R mutant protein. Cells were treated +/− SAHA and p65 acetylation determined by immunoblotting. Apoptosis was measured by caspase-3 activity, DNA fragmentation, and FACS analysis. Statistical significance was determined by Student’s t-test. Results: SAHA enhanced transcription of luciferase (p<0.003) and the endogenous genes IL-8, cIAP2, Bcl-XL, and Bfl/A1. These effects were attenuated by LY294002 (p<0.004). Acetylation of p65wt but not p65K310R was enhanced by SAHA. Expression of the p65K310R mutant protein reduced luciferase transcription relative to cells expressing p65wt (p<0.02). Treatment of NSCLC cells with both SAHA and LY294002 resulted in greater apoptosis compared to all other groups (p<0.002). Conclusions: SAHA dramatically enhanced NF-kB activity, in part, through acetylation of p65 and these effects were blocked by LY294002. Combined HDI and PI3K/Akt inhibition induced apoptosis in NSCLC and may represent a novel approach to the treatment of patients with NSCLC.