The cytokine TNFalpha is a strong inhibitor of renin gene expression in juxtaglomerular (JG) cells. We reported recently that TNFalpha suppresses renin transcription in the mouse JG cell line As4.1 acting through the transcription factor NFkappaB, which in turn targets a cAMP Responsive Element (CRE) in the mouse renin promoter. However, the exact mode of action of TNFalpha on the renin promoter bound NFkappaB remained unclear. Therefore, we aimed to study the mechanism of action of TNFalpha on NFkappaB transcriptional activity in As4.1 cells. Using a Gal4-dependent reporter gene system, we found that the transactivating capacity of the NFkB subunits p65 and cRel is massively reduced upon treatment with TNFalpha. The transactivation potential of p65 was more than 10-fold higher than that of cRel, suggesting that p65 essentailly mediates the effect of TNFalpha. Deletion analysis of the p65 molecule showed that TNFalpha targets its C-terminal half, which carries the transactivation domains. Further deletion studies demonstrated that the last 30 C-terminal amino acids of p65 are necessary and sufficient for the inhibitory effect of TNFalpha. These data suggest that NFkappaB-p65 plays central role in the downregulation of renin transcription by TNFalpha.