Fas-associated death domain protein (FADD) is an adaptor molecule linking death-receptors to caspase-8 in many cell types including cardiomyocytes (CM). While FADD has previously been reported to play an important role in cardiomyocyte apoptosis, the effect of FADD on cardiomyocyte NF-B signaling, a pro-inflammatory pathway, has not been delineated. To investigate the role of FADD in cardiomyocyte NF-B activation, we utilized adenoviral gene transfer of wild-type FADD and a truncation mutant lacking the death effector domain (FADD-DED) in rat cardiomyocytes in vitro. TNF- activated NF-B in cardiomyocytes as demonstrated by phosphorylation and degradation of IB-, enhanced nuclear p65 and NF-B DNA binding activity, as well as increased mRNA for the NF-B-dependent adhesion molecule, VCAM-1 (19 ±4.1 fold), as measured by quantitative RT-PCR. Gene transfer of FADD inhibited TNF--induced IB- phosphorylation, decreased p65 nuclear translocation and NF-B DNA binding activity, and reduced VCAM-1 transcript levels by 53-65%. Interestingly, FADD-DED exhibited a similar but weaker inhibitory effect on NF-B activation. The effects of FADD on NF-B were cell-type specific. FADD expression also inhibited TNF--mediated NF-B activation in human endothelial cells but not in rat pulmonary artery smooth muscle cells. In contrast, FADD expression actually activated NF-B in HEK293 cells. In cardiomyocytes, FADD inhibited NF-B activation as well as phosphorylation of IB- and IKK in response to cytokine stimulation or expression of the upstream kinases, NIK and IKK. These data demonstrate that FADD inhibits NF-B activation in cardiomyocytes and that this inhibition likely occurs at the level of phosphorylation and activation of IKK.